EP4363576A1 - Arn guides et polynucléotides modifiés - Google Patents

Arn guides et polynucléotides modifiés

Info

Publication number
EP4363576A1
EP4363576A1 EP22748526.5A EP22748526A EP4363576A1 EP 4363576 A1 EP4363576 A1 EP 4363576A1 EP 22748526 A EP22748526 A EP 22748526A EP 4363576 A1 EP4363576 A1 EP 4363576A1
Authority
EP
European Patent Office
Prior art keywords
relative
symmetric
bulge
composition
symmetric bulge
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22748526.5A
Other languages
German (de)
English (en)
Inventor
Richard Thomas SULLIVAN
Brian John BOOTH
Adrian Briggs
Yiannis SAVVA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shape Therapeutics Inc
Original Assignee
Shape Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shape Therapeutics Inc filed Critical Shape Therapeutics Inc
Publication of EP4363576A1 publication Critical patent/EP4363576A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense

Definitions

  • compositions that mediate RNA editing can be viable therapies for genetic diseases.
  • highly efficacious compositions that can maximize on-target RNA editing while minimizing off-target RNA editing are needed.
  • compositions that are capable of facilitating RNA editing are also needed.
  • engineered guide RNAs and composition comprising engineered guide RNAs, where: (a) the engineered guide RNA, upon hybridization to a c.l translation initiation sequence (TIS) of a target MAPT RNA, forms a guide-target RNA scaffold with the sequence of the target MAPT RNA, wherein the c.1 TIS is an AUG starting at the 18th nucleotide in Exon 1 of the target MAPT RNA; (b) formation of the guide-target RNA scaffold substantially forms one or more structural features selected from the group consisting of: a bulge, an internal loop, and a hairpin; (c) the structural feature is not present within the engineered guide RNA prior to the hybridization of the engineered guide RNA to the MAPT target RNA; and (d) upon hybridization of the engineered guide RNA to the c.l TIS of the target MAPT RNA, the engineered guide RNA facilitates RNA editing of one or more target adenosines in the c
  • the one or more structural features comprises: a first 6/6 symmetric internal loop at a position selected from the group consisting of: 34, 33, 32, 31, 30, 26, 25, 24, 23, and 22; relative to the target adenosine at position 0.
  • the first 6/6 symmetric internal loop is at position 34, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 478.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 479.
  • the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position - 5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 592.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 593.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/U mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 639.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/U mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 640.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 686.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 687.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 688.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position - 21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 695.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/U Wobble at position 6 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 696.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 8 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 714.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 8 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 715.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 722.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 723.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 3/3 symmetric bulge at position -13 relative to position 0, a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1059.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, an A/C mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1116.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, an A C mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1117.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1215
  • the first 6/6 symmetric internal loop is at position 32, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 438.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 439.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position - 10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 816.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position - 1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 817.
  • the first 6/6 symmetric internal loop is at position 31, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 162.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 163.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 929.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 930.
  • the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 538.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 539.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 4 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 604.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 4 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 605.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 619.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 620.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 677.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 678.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 684.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 685.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 720.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 721.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/A mismatch at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 733.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/A mismatch at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 734.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 763.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 764.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/G mismatch at position 8 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 775.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/G mismatch at position 8 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 776.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 795.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 796.
  • the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 464.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 465.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/A mismatch at position 11 relative to position 0, a G/U Wobble at position 18 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 583.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/A mismatch at position 11 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 584.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a U/U mismatch at position -11 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 589.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a G/U Wobble at position 18 relative to position 0, a G/U Wobble at position 20 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 610.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a G/U Wobble at position 18 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ED NO: 611.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 618.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/G mismatch at position -7 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 662.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 3 relative to position 0, a 2/2 symmetric bulge at position 7 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 664.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/C mismatch at position -7 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 2/2 symmetric bulge at position 10 relative to position 0, an A/C mismatch at position 19 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 672.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 675.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 676.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 699.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 700.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 718.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 719.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a 3/3 symmetric bulge at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 745.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 750.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/A mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 762.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a 3/3 symmetric bulge at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/U mismatch at position 5 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/G mismatch at position 17 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1099.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1120.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1213.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -22 relative to position 0, a 3/3 symmetric bulge mismatch at position -13 relative to position 0, a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1214.
  • the first 6/6 symmetric internal loop is at position 25, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -25 relative to position 0, a 3/3 symmetric bulge at position -16 relative to position 0, a 6/6 symmetric internal loop at position -4 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 88.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 903.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1179.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -4 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1212.
  • the first 6/6 symmetric internal loop is at position 24, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 118.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 119.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 270.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 271.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 403.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1081.
  • the first 6/6 symmetric internal loop is at position 23, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 401.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 402.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 458.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 459.
  • the first 6/6 symmetric internal loop is at position 22, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 34 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1135.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 34 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1136.
  • the one or more structural features comprises at least a first 6/6 symmetric internal loop and at least a second 6/6 symmetric loop.
  • the engineered guide RNA upon hybridization of the engineered guide RNA to the sequence of the target MAPT RNA, the engineered guide RNA facilitates RNA editing of one or more adenosines in the sequence of the target MAPT RNA by an RNA editing entity.
  • the RNA editing entity comprises ADAR1, ADAR2, ADAR3, or any combination thereof.
  • the engineered guide RNA is encoded by an engineered polynucleotide.
  • the engineered polynucleotide is comprised in or on a vector.
  • the vector is a viral vector, and wherein the engineered polynucleotide is encapsidated in the viral vector.
  • the viral vector is an adeno-associated viral (AAV) vector or a derivative thereof.
  • AAV adeno-associated viral
  • the viral vector is an adeno-associated viral (AAV) vector and wherein the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a derivative, a chimera, or a variant of any of these.
  • the AAV vector is a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, or any combination thereof.
  • the engineered guide RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 1215.
  • the composition is in unit dose form.
  • a composition described herein is for use in treatment of a disease or condition.
  • the disease or condition comprises a Tauopathy.
  • the disease or condition comprises Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy, autism, traumatic brain injury, or Dravet syndrome.
  • compositions comprising: (a) an engineered guide RNA described herein or a composition comprising an engineered guide RNA described herein; and (b) a pharmaceutically acceptable: excipient, carrier, or diluent.
  • the pharmaceutical composition is in unit dose form.
  • a pharmaceutical composition described herein is for use in treatment of a disease or condition.
  • the disease or condition comprises a Tauopathy.
  • the disease or condition comprises Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy, autism, traumatic brain injury, or Dravet syndrome.
  • AD Alzheimer’s disease
  • FDD frontotemporal dementia
  • PSP progressive supranuclear palsy
  • CBD corticobasal degeneration
  • chronic traumatic encephalopathy autism
  • traumatic brain injury or Dravet syndrome.
  • the disease or condition comprises a Tauopathy.
  • the disease or condition comprises Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy, autism, traumatic brain injury, or Dravet syndrome.
  • the subject is a human or a non-human animal.
  • the administering is sufficient to treat one or more symptoms of the disease of condition.
  • the one of more symptoms treated comprises a reduction in memory loss, a reduction in confusion, or any combination thereof, as compared to prior to the administering.
  • the administering is sufficient to reduce aggregation of tau protein, relative to: (a) a level of aggregation prior to the administering; (b) a level of accumulated aggregation in the subject in the absence of the administering; or (c) both.
  • the administering is sufficient to reduce an amount of: (a) neurofibrillary tangles; (b) neuronal cell death; (c) neuroinflammation; or (d) any combination thereof; relative to an amount prior to the administering.
  • the amount of phosphorylated tau protein is decreased in the subject after the administering, relative to an amount of phosphorylated tau protein prior to the administering.
  • a ratio of a 4R isoform of Tau to a 3R isoform of tau is increased upon administration, relative to a ratio prior to the administering.
  • Also disclosed herein are methods of editing a c.l translation initiation sequence (TIS) of a MAPT RNA the method comprising contacting the MAPT RNA with an engineered guide RNA described herein or a composition comprising an engineered guide RNA described herein, and an RNA editing entity, thereby editing the MAPT RNA.
  • TIS translation initiation sequence
  • at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the pre-mRNA transcripts of MAPT have at least one edit.
  • the editing of MAPT RNA facilitates a protein knockdown.
  • the protein knockdown comprises a reduction of at least 10%, relative to an amount of protein present prior to the contacting. In some embodiments, the protein knockdown comprises a reduction of from about 10% to about 25%, relative to an amount of protein present prior to the contacting. In some embodiments, the protein knockdown comprises a reduction of at least 50%, relative to an amount of protein present prior to the contacting. In some embodiments, the protein knockdown comprises a knockdown of tau. In some embodiments, the knockdown is measured in an in vitro assay. In some embodiments, the knockdown is measured in an in vivo assay. In some embodiments, the knockdown is measured in a human subject.
  • FIG. 1 shows a legend of various exemplary structural features present in guide-target RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA.
  • Example structural features shown include an 8/7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2/2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1/1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5/5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2/3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the
  • FIG. 2 is a plot showing, on the x-axis, the sequence similarity of the MAPT TIS- targeting engineered guide RNAs of the present disclosure to a canonical guide RNA design and, on the y-axis, the edited fraction by an ADAR2 enzyme.
  • FIG. 3 shows a schematic of the MAPT genomic landscape.
  • Engineered guide RNAs of the present disclosure can target any TIS in MAPT, including at the sites indicated as “ntl”, “nt31”, “nt91”, or a combination thereof “ntl” may also be referred to herein as “c.l” (coding nucleotide l) and is the 18 L h ⁇ o ⁇ Ecoh 1.
  • nt31 may also be referred to herein as “c.31” and is the 48 th nt of Exon 1.
  • nt91 may also be referred to herein as “c.91” and is the 108 th nt of Exon 1
  • RNA editing can refer to a process by which RNA is enzymatically modified post synthesis at specific nucleosides.
  • RNA editing can comprise any one of an insertion, deletion, or substitution of a nucleotide(s).
  • Examples of RNA editing include chemical modifications, such as pseudouridylation (the isomerization of uridine residues) and deamination (removal of an amine group from: cytidine to give rise to uridine, or C-to-U editing; or from adenosine to inosine, or A-to-I editing).
  • RNA editing can be used to correct mutations (e.g., correction of a missense mutation) in order to restore protein expression and to introduce mutations or edit coding or non-coding regions of RNA to inhibit RNA translation and effect protein knockdown.
  • RNA editing entity e.g., an adenosine Deaminase Acting on RNA (ADAR)
  • ADAR adenosine Deaminase Acting on RNA
  • engineered guide RNAs of the present disclosure can facilitate editing of a transcription initiation site (e.g.
  • ADARs can be enzymes that catalyze the chemical conversion of adenosines to inosines in RNA. Because the properties of inosine mimic those of guanosine (inosine will form two hydrogen bonds with cytosine, for example), inosine can be recognized as guanosine by the translational cellular machinery. “Adenosine-to-inosine (A-to-I) RNA editing”, therefore, effectively changes the primary sequence of RNA targets.
  • ADAR enzymes share a common domain architecture comprising a variable number of amino-terminal dsRNA binding domains (dsRBDs) and a single carboxy-terminal catalytic deaminase domain.
  • Human ADARs possess two or three dsRBDs.
  • Evidence suggests that ADARs can form homodimer as well as heterodimer with other ADARs when bound to double-stranded RNA, however it can be currently inconclusive if dimerization is needed for editing to occur.
  • the engineered guide RNAs disclosed herein can facilitate RNA editing by any of or any combination of the three human ADAR genes that have been identified (ADARs 1-3).
  • ADARs have a typical modular domain organization that includes at least two copies of a dsRNA binding domain (dsRBD; ADARlwith three dsRBDs; ADAR2 and ADAR3 each with two dsRBDs) in their N-terminal region followed by a C-terminal deaminase domain.
  • dsRBD dsRNA binding domain
  • ADARl with three dsRBDs
  • ADAR2 and ADAR3 each with two dsRBDs
  • the engineered guide RNAs (e.g. an engineered guide RNA of any one of SEQ ID NO: 2-1215 as recited in Table 1) of the present disclosure facilitate RNA editing (for example, of a MAPT c.1 transcription initiation site) by endogenous ADAR enzymes.
  • exogenous ADAR can be delivered alongside the engineered guide RNAs disclosed herein to facilitate RNA editing.
  • the ADAR is human AD AR1.
  • the ADAR is human ADAR2.
  • the ADAR is human ADAR3.
  • the ADAR is human ADARl, human ADAR2, human ADAR2, or any combination thereof.
  • the present disclosure provides engineered guide RNAs that facilitate edits at particular regions in a target RNA (e g., mRNA or pre-mRNA).
  • a target RNA e g., mRNA or pre-mRNA
  • the engineered guide RNAs disclosed herein can target a coding sequence or a non-coding sequence of an RNA.
  • a target region in a coding sequence of an RNA can be a translation initiation site (TIS).
  • TIS translation initiation site
  • the target region in a non-coding sequence of an RNA can be a polyadenylation (poly A) signal sequence.
  • the engineered guide RNAs of the present disclosure target the adenosine at a translation initiation site (TIS).
  • TIS translation initiation site
  • an engineered guide RNA of the present disclosure e.g. an engineered guide RNA of any one of SEQ ID NO: 2-1215 as recited in Table 1
  • the engineered guide RNAs facilitate ADAR-mediated RNA editing of the TIS (AUG) to GUG. This results in inhibition of RNA translation and, thereby, protein knockdown. Protein knockdown can also be referred to as reduced expression of wild-type protein.
  • Engineered guide RNAs of the present disclosure can target one or any combination of the TISs of MAPT.
  • engineered guide RNAs of the present disclosure target the TIS at the 18 th nt of Exon 1 (also referred to as the TIS at “c.l” for coding nucleotide 1”), such as an engineered guide RNA of any one of SEQ ID Nos: 2-1215.
  • engineered guide RNAs of the present disclosure target the TIS at the 48 th nt of Exon 1 (also referred to as the TIS at “c.31”).
  • engineered guide RNAs of the present disclosure target the TIS at the 108 th nt of Exon 1 (also referred to as the TIS at “c.91”). In some embodiments, engineered guide RNAs of the present disclosure target the TIS at the 6 th nt of Exon 5 (also referred to as the TIS at “c.379”). In some embodiments, engineered guide RNAs of the present disclosure target at least one TIS selected from the group consisting of c.l, c.31, c.91, and c.379.
  • engineered guide RNAs of the present disclosure target at least two TISs selected from the group consisting of c.l, c.31, c.91, and c.379. In some embodiments, engineered guide RNAs of the present disclosure target at least three TISs selected from the group consisting of c.l, c.31, c.91, and c.379. In some embodiments, engineered guide RNAs of the present disclosure target the c.1 TIS, the c.31 TIS, the c.91 TIS, and the c.379 TIS. In some embodiments, an engineered guide RNA of the present disclosure targets the c.l TIS.
  • an engineered guide RNA targeting the c.1 TIS is an engineered guide RNA having at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 1215.
  • the engineered guide RNAs of the present disclosure target one or more adenosines in the 3’ untranslated region (3’UTR).
  • an engineered guide RNA facilitates ADAR-mediated RNA editing of the one or more adenosines in the 3’UTR, thereby reducing mRNA export from the nucleus and inhibiting translation, thereby resulting in protein knockdown.
  • the engineered guide RNAs of the present disclosure target one or more adenosines in the 5’ untranslated region (5’ UTR). In some embodiments, an engineered guide RNA of the present disclosure can target a Kozak sequence of the 5’ UTR. In some embodiments, an engineered guide RNA of the present disclosure can target an internal ribosomal entry site (IRES) of the 5’ UTR. In some embodiments, an engineered guide RNA of the present disclosure can target an iron response element (IRE) of the 5’ UTR.
  • IRS internal ribosomal entry site
  • IRE iron response element
  • an engineered guide RNA facilitates ADAR-mediated RNA editing of one or more adenosines the 5’UTR (including one or more adenosines present in one or more structures of the 5’ UTR).
  • extensive or hyper editing of a plurality of adenosines can be facilitated via an engineered guide RNA of the present disclosure, which can result in ribosomal stalling of the mRNA transcript, thereby resulting in protein knockdown.
  • the engineered guide RNAs of the present disclosure target one or more adenosines in the polyA signal sequence.
  • an engineered guide RNA facilitates ADAR-mediated RNA editing of the one or more adenosines in the polyA signal sequence, thereby resulting in disruption of RNA processing and degradation of the target mRNA and, thereby, protein knockdown.
  • a target can have one or more polyA signal sequences.
  • one or more engineered guide RNAs, varying in their respective sequences, of the present disclosure can be multiplexed to target adenosines in the one or more polyA signal sequences.
  • engineered guide RNAs of the present disclosure targeting the canonical TIS (the c.l TIS) of MAPT can be multiplexed with one or more additional engineered guide RNAs targeting a different TIS of MAPT, such as the c.l 1 TIS, the c.31 TIS, or the c.91 TIS.
  • one or more engineered guide RNAs of the present disclosure targeting the canonical (c.l) TIS of MAPT may be multiplexed with or more engineered guide RNAs targeting a different sequence of MAPT, such as the 5’UTR region of MAPT (e.g., a Kozak sequence, an internal ribosomal entry site (IRES), or an iron response element (IRE) of the 5’ UTR).
  • the multiplexed engineered guide RNAs can be delivered together in the same viral vector or the each of the distinct engineered guide RNAs can be delivered together but in separate vectors.
  • the engineered guide RNAs of the present disclosure facilitated ADAR-mediated RNA editing of adenosines to inosines (read as guanosines by cellular machinery) in the polyA signal sequence, resulting in protein knockdown.
  • engineered guide RNAs e.g. an engineered guide RNA of any one of SEQ ID NO: 2-1215 as recited in Table 1
  • engineered polynucleotides encoding the same for site-specific, selective editing of a target RNA (for example, the c.1 TIS corresponding to the canonical TIS at nucleotide position 151 of the MAPT reference transcript of NM_001377265.1) via an RNA editing entity or a biologically active fragment thereof.
  • An engineered guide RNA of the present disclosure can comprise latent structures, such that when the engineered guide RNA is hybridized to the target RNA to form a guide-target RNA scaffold, at least a portion of the latent structure manifests as at least a portion of a structural feature as described herein.
  • An engineered guide RNA as described herein comprises a targeting domain with complementarity to a target RNA described herein.
  • a guide RNA can be engineered to site-specifically/selectively target and hybridize to a particular target RNA, thus facilitating editing of specific nucleotide in the target RNA via an RNA editing entity or a biologically active fragment thereof.
  • the targeting domain can include a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, the nucleotide opposes a base to be edited by the RNA editing entity or biologically active fragment thereof and does not base pair, or does not fully base pair, with the base to be edited.
  • This mismatch can help to localize editing of the RNA editing entity to the desired base of the target RNA.
  • Hybridization of the target RNA and the targeting domain of the guide RNA produces specific secondary structures in the guide-target RNA scaffold that manifest upon hybridization, which are referred to herein as “latent structures.”
  • Latent structures when manifested become structural features described herein, including mismatches, bulges, internal loops, and hairpins.
  • the presence of structural features described herein that are produced upon hybridization of the guide RNA with the target RNA configure the guide RNA to facilitate a specific, or selective, targeted edit of the target RNA via the RNA editing entity or biologically active fragment thereof.
  • the structural features in combination with the mismatch described above generally facilitate an increased amount of editing of a target adenosine, fewer off target edits, or both, as compared to a construct comprising the mismatch alone or a construct having perfect complementarity to a target RNA. Accordingly, rational design of latent structures in engineered guide RNAs of the present disclosure to produce specific structural features in a guide-target RNA scaffold can be a powerful tool to promote editing of the target RNA with high specificity, selectivity, and robust activity.
  • engineered guides and polynucleotides encoding the same as well as compositions comprising said engineered guide RNAs or said polynucleotides.
  • the term “engineered” in reference to a guide RNA or polynucleotide encoding the same refers to a non-naturally occurring guide RNA or polynucleotide encoding the same.
  • the present disclosure provides for engineered polynucleotides encoding engineered guide RNAs.
  • the engineered guide comprises RNA.
  • the engineered guide comprises DNA.
  • the engineered guide comprises modified RNA bases or unmodified RNA bases.
  • the engineered guide comprises modified DNA bases or unmodified DNA bases.
  • the engineered guide comprises both DNA and RNA bases.
  • the engineered guides provided herein comprise an engineered guide that can be configured, upon hybridization to a target RNA molecule, to form, at least in part, a guide-target RNA scaffold with at least a portion of the target RNA molecule, wherein the guide- target RNA scaffold comprises at least one structural feature, and wherein the guide-target RNA scaffold recruits an RNA editing entity and facilitates a chemical modification of a base of a nucleotide in the target RNA molecule by the RNA editing entity.
  • a target RNA of an engineered guide RNA of the present disclosure can be a pre-mRNA or mRNA.
  • the engineered guide RNA of the present disclosure hybridizes to a sequence of the target RNA.
  • part of the engineered guide RNA e.g., a targeting domain hybridizes to the sequence of the target RNA.
  • the part of the engineered guide RNA that hybridizes to the target RNA is of sufficient complementary to the sequence of the target RNA for hybridization to occur.
  • Engineered guide RNAs disclosed herein can be engineered in any way suitable for RNA editing.
  • an engineered guide RNA generally comprises at least a targeting sequence that allows it to hybridize to a region of a target RNA molecule (e.g. the c.l TIS corresponding to the canonical TIS at nucleotide position 151 of the MAPT reference transcript of NM_001377265.1).
  • a targeting sequence can also be referred to as a “targeting domain” or a “targeting region”.
  • a targeting domain of an engineered guide allows the engineered guide to target an RNA sequence through base pairing, such as Watson Crick base pairing.
  • the targeting sequence can be located at either the N-terminus or C-terminus of the engineered guide. In some cases, the targeting sequence can be located at both termini.
  • the targeting sequence can be of any length. In some cases, the targeting sequence can be at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • the targeting sequence can be no greater than about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • an engineered guide comprises a targeting sequence that can be from about 60 to about 500, from about 60 to about 200, from about 75 to about 100, from about 80 to about 200, from about 90 to about 120, or from about 95 to about 115 nucleotides in length.
  • an engineered guide RNA comprises a targeting sequence that can be about 100 nucleotides in length.
  • a targeting domain comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to a target RNA.
  • a targeting sequence comprises less than 100% complementarity to a target RNA sequence.
  • a targeting sequence and a region of a target RNA that can be bound by the targeting sequence can have a single base mismatch.
  • the targeting sequence can have sufficient complementarity to a target RNA to allow for hybridization of the targeting sequence to the target RNA.
  • the targeting sequence has a minimum antisense complementarity of about 50 nucleotides or more to the target RNA.
  • the targeting sequence has a minimum antisense complementarity of about 60 nucleotides or more to the target RNA.
  • the targeting sequence has a minimum antisense complementarity of about 70 nucleotides or more to the target RNA.
  • the targeting sequence has a minimum antisense complementarity of about 80 nucleotides or more to the target RNA.
  • the targeting sequence has a minimum antisense complementarity of about 90 nucleotides or more to the target RNA. In some embodiments, the targeting sequence has a minimum antisense complementarity of about 100 nucleotides or more to the target RNA. In some embodiments, antisense complementarity refers to non-contiguous stretches of sequence. In some embodiments, antisense complementarity refers to contiguous stretches of sequence.
  • a subject engineered guide RNA comprises a recruiting domain that recruits an RNA editing entity (e.g., ADAR), where in some instances, the recruiting domain is formed and present in the absence of binding to the target RNA.
  • a “recruiting domain” can be referred to herein as a “recruiting sequence” or a “recruiting region”.
  • a subject engineered guide can facilitate editing of a base of a nucleotide of in a target sequence of a target RNA that results in modulating the expression of a polypeptide encoded by the target RNA. In some instances, modulation can be decrease expression of the polypeptide.
  • an engineered guide can be configured to facilitate an editing of a base of a nucleotide or polynucleotide of a region of an RNA by an RNA editing entity (e.g., ADAR).
  • an engineered polynucleotide of the disclosure can recruit an RNA editing entity (e.g., ADAR).
  • RNA editing entity recruiting domains can be utilized.
  • a recruiting domain comprises: Glutamate ionotropic receptor AMPA type subunit 2 (GluR2), an Alu sequence, or, in the case of recruiting APOBEC, an APOBEC recruiting domain.
  • more than one recruiting domain can be included in an engineered guide of the disclosure.
  • the recruiting domain can be utilized to position the RNA editing entity to effectively react with a subject target RNA after the targeting sequence hybridizes to a target sequence of a target RNA.
  • a recruiting domain can allow for transient binding of the RNA editing entity to the engineered guide.
  • the recruiting domain allows for permanent binding of the RNA editing entity to the engineered guide.
  • a recruiting domain can be of any length. In some cases, a recruiting domain can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • a recruiting domain can be no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
  • a recruiting domain can be about 45 nucleotides in length. In some cases, at least a portion of a recruiting domain comprises at least 1 to about 75 nucleotides. In some cases, at least a portion of a recruiting domain comprises about 45 nucleotides to about 60 nucleotides.
  • a recruiting domain comprises a GluR2 sequence or functional fragment thereof.
  • a GluR2 sequence can be recognized by an RNA editing entity, such as an ADAR or biologically active fragment thereof.
  • a GluR2 sequence can be a non-naturally occurring sequence.
  • a GluR2 sequence can be modified, for example for enhanced recruitment.
  • a GluR2 sequence can comprise a portion of a naturally occurring GluR2 sequence and a synthetic sequence.
  • a recruiting domain comprises a GluR2 sequence, or a sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity and/or length to: GUGGAAUAGUAUAACAAUAUGCUAAAUGUUGUUAUAGUAUCCCAC (SEQ ID NO:
  • a recruiting domain can comprise at least about 80% sequence homology to at least about 10, 15, 20, 25, or 30 nucleotides of SEQ ID NO: 1. In some examples, a recruiting domain can comprise at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence homology and/or length to SEQ ID NO: 1.
  • a recruiting domain comprises an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) domain.
  • APOBEC catalytic polypeptide-like
  • an APOBEC domain can comprise a non-naturally occurring sequence or naturally occurring sequence.
  • an APOBEC-domain-encoding sequence can comprise a modified portion.
  • an APOBEC-domain-encoding sequence can comprise a portion of a naturally occurring APOBEC- domain-encoding-sequence.
  • a recruiting domain can be from an Alu domain.
  • recruiting domains can be found in an engineered guide of the present disclosure. In some examples, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 recruiting domains can be included in an engineered guide.
  • recruiting domains can be located at any position of engineered guide RNAs. In some cases, a recruiting domain can be on an N-terminus, middle, or C-terminus of an engineered guide RNA.
  • a recruiting domain can be upstream or downstream of a targeting sequence. In some cases, a recruiting domain flanks a targeting sequence of a subject guide.
  • a recruiting sequence can comprise all ribonucleotides or deoxyribonucleotides, although a recruiting domain comprising both ribo- and deoxyribonucleotides can in some cases not be excluded.
  • an engineered guide disclosed herein useful for facilitating editing of a target RNA by an RNA editing entity can be an engineered latent guide RNA.
  • An “engineered latent guide RNA” refers to an engineered guide RNA that comprises latent structure.
  • “Latent structure” refers to a structural feature that substantially forms upon hybridization of a guide RNA to a target RNA.
  • the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA.
  • the structural feature is formed and the latent structure provided in the guide RNA is, thus, unmasked.
  • a double stranded RNA (dsRNA) substrate is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA (for example, a MAPT c.1 TIS).
  • a target RNA for example, a MAPT c.1 TIS.
  • the resulting dsRNA substrate is also referred to herein as a “guide-target RNA scaffold.”
  • FIG. 1 shows a legend of various exemplary structural features present in guide-target RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA.
  • Example structural features shown include an 8/7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2/2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1/1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5/5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2/3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the
  • the number of participating nucleotides in a given structural feature is indicated as the nucleotides on the target RNA side over nucleotides on the guide RNA side. Also shown in this legend is a key to the positional annotation of each figure.
  • the target nucleotide to be edited is designated as the 0 position.
  • Downstream (3’) of the target nucleotide to be edited each nucleotide is counted in increments of +1.
  • Upstream (5’) of the target nucleotide to be edited each nucleotide is counted in increments of -1.
  • the example 2/2 symmetric bulge in this legend is at the +12 to +13 position in the guide-target RNA scaffold.
  • the 2/3 asymmetric bulge in this legend is at the -36 to-37 position in the guide-target RNA scaffold.
  • positional annotation is provided with respect to the target nucleotide to be edited and on the target RNA side of the guide-target RNA scaffold.
  • the structural feature extends from that position away from position 0 (target nucleotide to be edited).
  • a latent guide RNA is annotated herein as forming a 2/3 asymmetric bulge at position -36, then the 2/3 asymmetric bulge forms from -36 position to the -37 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold.
  • a latent guide RNA is annotated herein as forming a 2/2 symmetric bulge at position +12, then the 2/2 symmetric bulge forms from the +12 to the +13 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold.
  • the engineered guides disclosed herein lack a recruiting region and recruitment of the RNA editing entity can be effectuated by structural features of the guide-target RNA scaffold formed by hybridization of the engineered guide RNA and the target RNA.
  • the engineered guide when present in an aqueous solution and not bound to the target RNA molecule, does not comprise structural features that recruit the RNA editing entity (e g., ADAR).
  • the engineered guide RNA upon hybridization to a target RNA, form with the target RNA molecule, one or more structural features that recruits an RNA editing entity (e.g., ADAR) [0040]
  • an engineered guide RNA can be still capable of associating with a subject RNA editing entity (e g., ADAR) to facilitate editing of a target RNA and/or modulate expression of a polypeptide encoded by a subject target RNA.
  • This can be achieved through structural features formed in the guide-target RNA scaffold formed upon hybridization of the engineered guide RNA and the target RNA.
  • Structural features can comprise any one of a: mismatch, symmetrical bulge, asymmetrical bulge, symmetrical internal loop, asymmetrical internal loop, hairpins, wobble base pairs, or any combination thereof.
  • features include a mismatch, a bulge (symmetrical bulge or asymmetrical bulge), an internal loop (symmetrical internal loop or asymmetrical internal loop), or a hairpin (a recruiting hairpin or a non-recruiting hairpin).
  • Engineered guide RNAs of the present disclosure can have from 1 to 50 features.
  • Engineered guide RNAs of the present disclosure can have from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45 to 50, from 5 to 20, from 1 to 3, from 4 to 5, from 2 to 10, from 20 to 40, from 10 to 40, from 20 to 50, from 30 to 50, from 4 to 7, or from 8 to 10 features.
  • structural features e g., mismatches, bulges, internal loops
  • structural features are not formed from latent structures and are, instead, pre-formed structures (e g., a GluR2 recruitment hairpin or a hairpin from U7 snRNA).
  • a guide-target RNA scaffold is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA.
  • a mismatch refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNA scaffold.
  • a mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceeds 1, the resulting structure is no longer considered a mismatch, but rather, is considered a bulge or an internal loop, depending on the size of the structural feature.
  • a mismatch is an A/C mismatch.
  • An A/C mismatch can comprise a C in an engineered guide RNA of the present disclosure opposite an A in a target RNA.
  • An A/C mismatch can comprise an A in an engineered guide RNA of the present disclosure opposite a C in a target RNA.
  • a G/G mismatch can comprise a G in an engineered guide RNA of the present disclosure opposite a G in a target RNA.
  • a mismatch positioned 5’ of the edit site can facilitate base flipping of the target A to be edited.
  • a mismatch can also help confer sequence specificity.
  • a mismatch can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • a structural feature comprises a wobble base.
  • a wobble base pair refers to two bases that weakly base pair.
  • a wobble base pair of the present disclosure can refer to a G paired with a U.
  • a wobble base pair can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • a structural feature can be a hairpin.
  • a hairpin includes an RNA duplex wherein a portion of a single RNA strand has folded in upon itself to form the RNA duplex. The portion of the single RNA strand folds upon itself due to having nucleotide sequences that base pair to each other, where the nucleotide sequences are separated by an intervening sequence that does not base pair with itself, thus forming a base-paired portion and non-base paired, intervening loop portion.
  • a hairpin can have from 10 to 500 nucleotides in length of the entire duplex structure.
  • the loop portion of a hairpin can be from 3 to 15 nucleotides long.
  • a hairpin can be present in any of the engineered guide RNAs disclosed herein.
  • the engineered guide RNAs disclosed herein can have from 1 to 10 hairpins. In some embodiments, the engineered guide RNAs disclosed herein have 1 hairpin. In some embodiments, the engineered guide RNAs disclosed herein have 2 hairpins.
  • a hairpin can include a recruitment hairpin or a non-recruitment hairpin. A hairpin can be located anywhere within the engineered guide RNAs of the present disclosure.
  • one or more hairpins is proximal to or present at the 3 end of an engineered guide RNA of the present disclosure, proximal to or at the 5 end of an engineered guide RNA of the present disclosure, proximal to or within the targeting domain of the engineered guide RNAs of the present disclosure, or any combination thereof.
  • a structural feature comprises a non-recruitment hairpin.
  • a non recruitment hairpin does not have a primary function of recruiting an RNA editing entity.
  • a non-recruitment hairpin in some instances, does not recruit an RNA editing entity.
  • a non-recruitment hairpin has a dissociation constant for binding to an RNA editing entity under physiological conditions that is insufficient for binding.
  • a non-recruitment hairpin has a dissociation constant for binding an RNA editing entity at 25 °C that is greater than about 1 mM, 10 mM, 100 mM, or 1 M, as determined in an in vitro assay.
  • a non-recruitment hairpin can exhibit functionality that improves localization of the engineered guide RNA to the target RNA.
  • the non-recruitment hairpin improves nuclear retention.
  • the non-recruitment hairpin comprises a hairpin from U7 snRNA.
  • a non-recruitment hairpin such as a hairpin from U7 snRNA is a pre-formed structural feature that can be present in constructs comprising engineered guide RNA constructs, not a structural feature formed by latent structure provided in an engineered latent guide RNA.
  • a hairpin of the present disclosure can be of any length.
  • a hairpin can be from about 10-500 or more nucleotides.
  • a hairpin can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
  • a hairpin can also comprise 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 160, 10 to 170, 10 to 180, 10 to
  • a guide-target RNA scaffold is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA.
  • a bulge refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand.
  • a bulge can change the secondary or tertiary structure of the guide-target RNA scaffold.
  • a bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold.
  • a bulge does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair - a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a mismatch.
  • the resulting structure is no longer considered a bulge, but rather, is considered an internal loop.
  • the guide-target RNA scaffold of the present disclosure has 2 bulges.
  • the guide-target RNA scaffold of the present disclosure has 3 bulges. In some embodiments, the guide-target RNA scaffold of the present disclosure has 4 bulges.
  • a bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • the presence of a bulge in a guide-target RNA scaffold can position or can help to position ADAR to selectively edit the target A in the target RNA and reduce off-target editing of non-target A(s) in the target RNA.
  • the presence of a bulge in a guide-target RNA scaffold can recruit or help recruit additional amounts of ADAR.
  • Bulges in guide-target RNA scaffolds disclosed herein can recruit other proteins, such as other RNA editing entities.
  • a bulge positioned 5’ of the edit site can facilitate base-flipping of the target A to be edited.
  • a bulge can also help confer sequence specificity for the A of the target RNA to be edited, relative to other A(s) present in the target RNA.
  • a bulge can help direct ADAR editing by constraining it in an orientation that yields selective editing of the target A.
  • a guide-target RNA scaffold is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA.
  • a bulge can be a symmetrical bulge or an asymmetrical bulge.
  • a symmetrical bulge is formed when the same number of nucleotides is present on each side of the bulge.
  • a symmetrical bulge in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold.
  • a symmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 2 nucleotides on the target RNA side of the guide- target RNA scaffold.
  • a symmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 3 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical bulge of the present disclosure can be formed by 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 4 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • a guide-target RNA scaffold is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA.
  • a bulge can be a symmetrical bulge or an asymmetrical bulge.
  • An asymmetrical bulge is formed when a different number of nucleotides is present on each side of the bulge.
  • an asymmetrical bulge in a guide-target RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 1 nucleotide on the target RNA side of the guide- target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • an asymmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • an asymmetric bulge can be a 1/0 asymmetric bulge.
  • a 1/0 asymmetric bulge can be a U deletion.
  • a “U deletion” refers to a 1/0 asymmetric bulge in which a U nucleotide of an engineered guide RNA that would be situated opposite a non-target A of a target RNA in the guide-target RNA scaffold is deleted from the engineered guide RNA.
  • a 1/0 asymmetric bulge comprising a U deletion can reduce editing of the non-target A, relative to a comparable guide RNA lacking the U deletion.
  • a structural feature can be an internal loop.
  • an internal loop refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, has 5 nucleotides or more. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a bulge or a mismatch, depending on the size of the structural feature.
  • An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. Internal loops present in the vicinity of the edit site can help with base flipping of the target A in the target RNA to be edited
  • One side of the internal loop can be formed by from 5 to 150 nucleotides.
  • One side of the internal loop can be formed by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,
  • One side of the internal loop can be formed by 5 nucleotides.
  • One side of the internal loop can be formed by 10 nucleotides.
  • One side of the internal loop can be formed by 15 nucleotides.
  • One side of the internal loop can be formed by 20 nucleotides.
  • One side of the internal loop can be formed by 25 nucleotides.
  • One side of the internal loop can be formed by 30 nucleotides.
  • One side of the internal loop can be formed by 35 nucleotides.
  • One side of the internal loop can be formed by 40 nucleotides.
  • One side of the internal loop can be formed by 45 nucleotides.
  • One side of the internal loop can be formed by 50 nucleotides.
  • One side of the internal loop can be formed by 55 nucleotides.
  • One side of the internal loop can be formed by 60 nucleotides.
  • One side of the internal loop can be formed by 65 nucleotides.
  • One side of the internal loop can be formed by 70 nucleotides.
  • One side of the internal loop can be formed by 75 nucleotides.
  • One side of the internal loop can be formed by 80 nucleotides.
  • One side of the internal loop can be formed by 85 nucleotides.
  • One side of the internal loop can be formed by 90 nucleotides.
  • One side of the internal loop can be formed by 95 nucleotides.
  • One side of the internal loop can be formed by 100 nucleotides.
  • One side of the internal loop can be formed by 110 nucleotides.
  • One side of the internal loop can be formed by 120 nucleotides.
  • One side of the internal loop can be formed by 130 nucleotides.
  • One side of the internal loop can be formed by 140 nucleotides.
  • One side of the internal loop can be formed by 150 nucleotides.
  • One side of the internal loop can be formed by 200 nucleotides.
  • One side of the internal loop can be formed by 250 nucleotides.
  • One side of the internal loop can be formed by 300 nucleotides.
  • One side of the internal loop can be formed by 350 nucleotides.
  • One side of the internal loop can be formed by 400 nucleotides.
  • an internal loop can be formed by 450 nucleotides.
  • One side of the internal loop can be formed by 500 nucleotides.
  • One side of the internal loop can be formed by 600 nucleotides.
  • One side of the internal loop can be formed by 700 nucleotides.
  • One side of the internal loop can be formed by 800 nucleotides.
  • One side of the internal loop can be formed by 900 nucleotides.
  • One side of the internal loop can be formed by 1000 nucleotides.
  • an internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • An internal loop can be a symmetrical internal loop or an asymmetrical internal loop.
  • a symmetrical internal loop is formed when the same number of nucleotides is present on each side of the internal loop.
  • a symmetrical internal loop in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 5 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 6 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 7 nucleotides on the target RNA side of the guide- target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 8 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 9 nucleotides on the target RNA side of the guide- target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 10 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 15 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 15 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 20 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 20 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 30 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 30 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 40 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 40 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 50 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 60 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 60 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 70 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 70 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 80 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 80 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 90 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 90 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 100 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 110 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 110 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 120 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 120 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 130 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 130 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 140 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold target and 140 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 150 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 200 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 250 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 250 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 300 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 350 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 350 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 400 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 450 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 450 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 500 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 600 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold target and 600 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 700 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 700 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 800 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 800 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 900 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 900 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 1000 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • a symmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • An asymmetrical internal loop is formed when a different number of nucleotides is present on each side of the internal loop.
  • an asymmetrical internal loop in a guide- target RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by from 5 to 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and from 5 to 150 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the number of nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by from 5 to 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and from 5 to 1000 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the number of nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides on the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide- target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide- target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide- target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide- target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide- target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide- target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide- target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide- target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide- target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide- target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide- target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide- target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide- target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide- target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide- target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide- target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide- target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide- target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide- target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide- target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide- target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide- target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide- target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide- target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide- target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide- target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide- target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide- target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide- target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide- target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide- target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold.
  • an asymmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
  • a “base paired (bp) region” refers to a region of the guide-target RNA scaffold in which bases in the guide RNA are paired with opposing bases in the target RNA.
  • Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to the other end of the guide-target RNA scaffold.
  • Base paired regions can extend between two structural features.
  • Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to a structural feature.
  • Base paired regions can extend from a structural feature to the other end of the guide-target RNA scaffold.
  • a base paired region has from 1 bp to 100 bp, from 1 bp to 90 bp, from 1 bp to 80 bp, from 1 bp to 70 bp, from 1 bp to 60 bp, from 1 bp to 50 bp, from 1 bp to 45 bp, from 1 bp to 40 bp, from 1 bp to 35 bp, from 1 bp to 30 bp, from 1 bp to 25 bp, from 1 bp to 20 bp, from 1 bp to 15 bp, from 1 bp to 10 bp, from 1 bp to 5 bp, from 5 bp to 10 bp, from 5 bp to 20 bp, from 10 bp to 20 bp, from 10 bp to 50 bp, from 5 bp to 50 bp, at least 1 bp, at least 2 bp, at least 3 bp, at least
  • engineered guide RNAs for example, an engineered guide RNA of any one of SEQ ID NO: 2-1215 as recited in Table 1 that target a sequence of a MAPT target RNA (for example, the c.l TIS corresponding to the canonical TIS at nucleotide position 151 of the MAPT reference transcript of NM_001377265.1)
  • an engineered guide RNA comprises one or more structural features that manifest as latent structures which result in editing of a target adenosine (defined as position 0) in a c.l TIS target sequence of a MAPT RNA.
  • the one or more structural features comprises a first 6/6 symmetric internal loop and a second symmetric 6/6 internal loop.
  • the one or more structural features comprises: a first 6/6 symmetric internal loop at a position selected from the group consisting of: 34, 33, 32, 31, 30, 26, 25, 24,
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 34, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 478.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 478.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 34, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 479.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 479.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 592. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 592
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 593.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 593.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/U mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 9
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/U mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 640.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 640.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 686.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 686.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 687.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 687.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 688.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 695.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 695.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/U Wobble at position 6 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 696. In some cases, an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 696.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 8 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 714.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 714.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 8 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 715.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 715.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 722.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 722.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 723.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 723
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 3/3 symmetric bulge at position -13 relative to position 0, a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1059.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1059.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, an A/C mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO:
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1116.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, an A/C mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a C U mismatch at position 14 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1117.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1117.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1117.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1215.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 32, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 438.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 438.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 32, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 439.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 439.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 32, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 816.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 32, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 817. In some cases, an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 817.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 31, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 162.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 162.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 31, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 163.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 163.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 31, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 929. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 929.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 31, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 930. In some cases, an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 930.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 538.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 538.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 539.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 539.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 4 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 604. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 604.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 4 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 605.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 605.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 619.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 619.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 620.
  • an engineered guide RNA of the present disclosure that targets a c
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 677. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 677.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 678.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 678.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 684. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 684.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 685.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 685.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 720.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 720.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 721.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 721.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/A mismatch at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 733. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 733
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/A mismatch at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising S
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 763.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 763.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 764. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 764.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/G mismatch at position 8 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 775. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 775.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/G mismatch at position 8 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 776.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 776.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 795.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 795.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 796.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 796.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 464. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 464.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 465.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/A mismatch at position 11 relative to position 0, a G/U Wobble at position 18 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 583. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 583.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/A mismatch at position 11 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 584.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 584.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a U/U mismatch at position -11 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 589.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 589.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a G/U Wobble at position 18 relative to position 0, a G/U Wobble at position 20 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 610.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 610.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a G/U Wobble at position 18 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 611.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 611.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 618. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 618.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/G mismatch at position -7 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 662. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 662.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G U Wobble at position 3 relative to position 0, a 2/2 symmetric bulge at position 7 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 664. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 664.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/C mismatch at position -7 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 2/2 symmetric bulge at position 10 relative to position 0, an A/C mismatch at position 19 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 672. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 672.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 675. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 675.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 676.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 676.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 699.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 699.
  • sequence of a MAPT RNA has a sequence of SEQ ID NO: 676.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 700.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 700.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 718. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 718.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 719.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 719.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a 3/3 symmetric bulge at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 745.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 745.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 750.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 750.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/A mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 762. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 762.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a 3/3 symmetric bulge at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/U mismatch at position 5 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/G mismatch at position 17 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1099.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1099.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO:
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1120.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1213.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1213.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -22 relative to position 0, a 3/3 symmetric bulge mismatch at position -13 relative to position 0, a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1214.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1214.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 25, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -25 relative to position 0, a 3/3 symmetric bulge at position -16 relative to position 0, a 6/6 symmetric internal loop at position -4 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 88. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 88.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 25, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 903.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 903.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 25, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1179. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1179.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 25, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -4 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1212. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1212.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 24, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 118. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 118.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 24, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 119.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 119.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 24, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 270.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 270.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 24, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 271.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 24, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 403.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 403.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 24, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1081.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1081.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 23, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 401.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 401
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 23, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 402.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 402.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 23, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 458. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 458
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 23, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 459.
  • an engineered guide RNA of the present disclosure that targets a c.l TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 459.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 22, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 34 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO:
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1135.
  • the one or more structural features comprises the first 6/6 symmetric internal loop is at position 22, relative to the target adenosine at position 0.
  • the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 34 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to a guide RNA comprising SEQ ID NO: 1136. In some cases, an engineered guide RNA of the present disclosure that targets a c.1 TIS target sequence of a MAPT RNA has a sequence of SEQ ID NO: 1136.
  • an engineered guide RNA described herein can be circular.
  • an engineered guide RNA described herein can comprise a U7, an SmOPT sequence, or a combination of both sequences.
  • an engineered guide RNA can be circularized. In some cases, an engineered guide RNA provided herein can be circularized or in a circular configuration. In some aspects, an at least partially circular guide RNA lacks a 5’ hydroxyl or a 3’ hydroxyl. In some embodiments, a circular engineered guide RNA can comprise a guide RNA from any one of SEQ ID NOs: 2-1215 as recited in Table 1 that target MAPT c.l TIS.
  • an engineered guide RNA can comprise a backbone comprising a plurality of sugar and phosphate moieties covalently linked together.
  • a backbone of an engineered guide RNA can comprise a phosphodiester bond linkage between a first hydroxyl group in a phosphate group on a 5’ carbon of a deoxyribose in DNA or ribose in RNA and a second hydroxyl group on a 3’ carbon of a deoxyribose in DNA or ribose in RNA.
  • a backbone of an engineered guide RNA can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to a solvent. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to nucleases. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to hydrolytic enzymes.
  • a backbone of an engineered guide can be represented as a polynucleotide sequence in a circular 2-dimensional format with one nucleotide after the other. In some instances, a backbone of an engineered guide can be represented as a polynucleotide sequence in a looped 2-dimensional format with one nucleotide after the other.
  • a 5’ hydroxyl, a 3’ hydroxyl, or both can be joined through a phosphorus-oxygen bond. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be modified into a phosphoester with a phosphorus-containing moiety.
  • an engineered guide can comprise a circular structure.
  • An engineered polynucleotide can be circularized from a precursor engineered polynucleotide.
  • a precursor engineered polynucleotide can be a precursor engineered linear polynucleotide.
  • a precursor engineered linear polynucleotide can be a precursor for a circular engineered guide RNA.
  • a precursor engineered linear polynucleotide can be a linear mRNA transcribed from a plasmid, which can be configured to circularize within a cell using the techniques described herein.
  • a precursor engineered linear polynucleotide can be constructed with domains such as a ribozyme domain and a ligation domain that allow for circularization when inserted into a cell.
  • a ribozyme domain can include a domain that is capable of cleaving the linear precursor RNA at specific sites ( e.g ., adjacent to the ligation domain).
  • a precursor engineered linear polynucleotide can comprise, from 5’ to 3’: a 5’ ribozyme domain, a 5’ ligation domain, a circularized region, a 3’ ligation domain, and a 3’ ribozyme domain.
  • a circularized region can comprise a guide RNA described herein.
  • the precursor polynucleotide can be specifically processed at both sites by the 5’ and the 3’ ribozymes, respectively, to free exposed ends on the 5’ and 3’ ligation domains.
  • the free exposed ends can be ligation competent, such that the ends can be ligated to form a mature circularized structure.
  • the free ends can include a 5 ’-OH and a 2’,
  • RNA 3 ’-cyclic phosphate that are ligated via RNA ligation in the cell.
  • the linear polynucleotide with the ligation and ribozyme domains can be transfected into a cell where it can circularize via endogenous cellular enzymes.
  • a polynucleotide can encode an engineered guide RNA comprising the ribozyme and ligation domains described herein, which can circularize within a cell. Circular guide RNAs are described in PCT/US2021/034301, which is incorporated by reference in its entirety.
  • An engineered polynucleotide as described herein can include spacer domains.
  • a spacer domain can refer to a domain that provides space between other domains.
  • a spacer domain can be used to between a region to be circularized and flanking ligation sequences to increase the overall size of the mature circularized guide RNA.
  • the region to be circularized includes a targeting domain as described herein that is configured to associate to a target sequence, the addition of spacers can provide improvements (e.g .
  • a precursor engineered polynucleotide or a circular engineered guide can comprise, in order of 5’ to 3’: a first ribozyme domain; a first ligation domain; a first spacer domain; a targeting domain that can be at least partially complementary to a target RNA, a second spacer domain, a second ligation domain, and a second ribozyme domain.
  • the first spacer domain, the second spacer domain, or both are configured to not bind to the target RNA when the targeting domain binds to the target RNA.
  • compositions and methods of the present disclosure provide engineered polynucleotides encoding for guide RNAs that are operably linked to a portion of a small nuclear ribonucleic acid (snRNA) sequence.
  • the engineered polynucleotide can include at least a portion of a small nuclear ribonucleic acid (snRNA) sequence.
  • snRNA small nuclear ribonucleic acid
  • snRNA is a class of small RNA molecules found within the nucleus of eukaryotic cells. They are involved in a variety of important processes such as RNA splicing (removal of introns from pre-mRNA), regulation of transcription factors (7SK RNA) or RNA polymerase II (B2 RNA), and maintaining the telomeres. They are always associated with specific proteins, and the resulting RNA-protein complexes are referred to as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps.
  • snRNAs which are denominated U1 , U2, U3, U4, U5, U6, U7, U8, U9, and U10.
  • Wild-type U7 snRNA includes a stem-loop structure, the U7-specific Sm sequence, and a sequence antisense to the 3' end of histone pre-mRNA.
  • U7 comprises a sequence antisense to the 3' end of histone pre-mRNA.
  • this sequence is replaced by a targeting sequence that is antisense to another target pre-mRNA, U7 is redirected to the new target pre-mRNA. Accordingly, the stable expression of modified U7 snRNAs containing the SmOPT domain and a targeting antisense sequence has resulted in specific alteration of mRNA splicing.
  • AAV-2/1 based vectors expressing an appropriately modified murine U7 gene along with its natural promoter and 3' elements have enabled high efficiency gene transfer into the skeletal muscle and complete dystrophin rescue by covering and skipping mouse Dmd exon 23, the engineered polynucleotides as described herein (whether directly administered or administered via, for example, AAV vectors) can facilitate editing of target RNA by a deaminase.
  • the engineered polynucleotide can comprise at least in part an snRNA sequence.
  • the snRNA sequence can be Ul, U2, U3, U4, U5, U6, U7, U8, U9, or a U10 snRNA sequence.
  • an engineered polynucleotide that comprises at least a portion of an snRNA sequence e g. an snRNA promoter, an snRNA hairpin, and the like
  • an engineered polynucleotide that comprises at least a portion of an snRNA sequence can have superior properties for treating or preventing a disease or condition, relative to a comparable polynucleotide lacking such features.
  • an engineered polynucleotide that comprises at least a portion of an snRNA sequence can facilitate exon skipping of an exon at a greater efficiency than a comparable polynucleotide lacking such features
  • an engineered polynucleotide that comprises at least a portion of an snRNA sequence can facilitate an editing of a base of a nucleotide in a target RNA (e g. a pre-mRNA or a mature RNA) at a greater efficiency than a comparable polynucleotide lacking such features.
  • a target RNA e g. a pre-mRNA or a mature RNA
  • RNAs comprising (a) an engineered guide RNA as described herein, and (b) a U7 snRNA hairpin sequence, a SmOPT sequence, or a combination thereof.
  • the U7 hairpin comprises a human U7 Hairpin sequence, or a mouse U7 hairpin sequence.
  • a human U7 hairpin sequence comprises TAGGCTTTCTGGCTTTTTTTACCGGAAAGCCCCT (SEQ ID NO: 1216 or RNA: UAGGCUUUCUGGCUUUUUACCGGAAAGCCCCU (SEQ ID NO: 1217).
  • a mouse U7 hairpin sequence comprises CAGGTTTTCTGACTTCGGTCGGAAAACCCCT (SEQ ID NO: 1218 or RNA: CAGGUUUUCUGACUUCGGUCGGAAAACCCCU SEQ ID NO: 1219).
  • the SmOPT sequence has a sequence of AATTTTTGGAG (SEQ ID NO: 1220 or RNA: A AUUUUU GGAG SEQ ID NO: 1221).
  • a guide RNA from any one of SEQ ID NOs: 2-1215 as recited in Table 1 that target MAPT c.l TIS can comprise a guide RNA comprising a U7 hairpin sequence (e.g., a human or a mouse U7 hairpin sequence), an SmOPT sequence, or a combination thereof.
  • a combination of a U7 hairpin sequence and a SmOPT sequence can comprise a SmOPT U7 hairpin sequence, wherein the SmOPT sequence is linked to the U7 sequence.
  • a U7 hairpin sequence, an SmOPT sequence, or a combination thereof is downstream (e.g., 3’) of the engineered guide RNA disclosed herein.
  • a promoter for driving the expression of a guide RNA disclosed herein.
  • the promoters for driving expression can be 5’ to the guide RNA sequence disclosed herein.
  • a promoter can comprise a U1 promoter, a U7 promoter, a U6 promoter or any combination thereof.
  • a promoter can comprise a CMV promoter.
  • a U7 promoter, or a U6 promoter can be a mouse U7 promoter, or a mouse U6 promoter.
  • a U1 promoter, a U7 promoter, or a U6 promoter can be a human U1 promoter, a human U7 promoter, or a human U6 promoter.
  • a human U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:
  • a mouse U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:
  • a human U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: TTAACAACAACGAAGGGGCTGTGACTGGCTGCTTTCTCAACCAATCAGCACCGAACT CATTTGCATGGGCTGAGAACAAATGTTCGCGAACTCTAGAAATGAATGACTTAAGT AAGTTCCTTAGAATATTATTTTTCCTACTGAAAGTTACCACATGCGTCGTTGTTTATA CAGTAATAGGAACAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  • a human U1 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:
  • a CMV promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to:
  • the present disclosure provides for compositions of engineered guide RNAs or engineered polynucleotides encoding guide RNAs and methods of use thereof, such as methods of treatment.
  • the engineered polynucleotides of the present disclosure encode guide RNAs targeting a coding sequence of an RNA (e.g., a TIS such as the c.l TIS, the c.31 TIS, the c.91 TIS, or the c.379 TIS of MAPT).
  • a TIS such as the c.l TIS, the c.31 TIS, the c.91 TIS, or the c.379 TIS of MAPT.
  • the engineered polynucleotides of the present disclosure encode guide RNAs target the c.l TIS and have at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 2 - SEQ ID NO: 1215.
  • the engineered polynucleotides of the present disclosure encode guide RNAs targeting a non-coding sequence of an RNA (e.g., a polyA sequence).
  • the present disclosure provides compositions of one or more than one engineered polynucleotides encoding more than one engineered guide RNAs targeting the TIS, the polyA sequence, or any other part of a coding sequence or non-coding sequence.
  • the engineered guide RNAs disclosed herein facilitate ADAR-mediated RNA editing of adenosines in the TIS, the polyA sequence, any part of a coding sequence of an RNA, any part of a non coding sequence of an RNA, or any combination thereof.
  • the present disclosure provides for engineered guide RNAs that facilitate MAPT RNA editing when contacted with MAPT RNA to knockdown expression of Tau protein. Knockdown via an engineered guide RNA of the present disclosure result in a reduction of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of Tau protein, relative to an amount prior to contacting the engineered guide RNA with the MAPT RNA.
  • Tau pathology can be a key driver of a broad spectrum of neurodegenerative diseases, collectively known as Tauopathies.
  • diseases where Tau can play a primary role include, but are not limited to, Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and chronic traumatic encephalopathy.
  • AD Alzheimer’s disease
  • FDD frontotemporal dementia
  • PSP progressive supranuclear palsy
  • CBD corticobasal degeneration
  • Tauopathies are characterized by the intracellular accumulation of neurofibrillary tangles (NFTs) composed of aggregated, misfolded Tau (MAPT gene).
  • NFTs neurofibrillary tangles
  • engineered guide RNAs of the present disclosure targeting MAPT RNA for ADAR-mediated editing to knockdown Tau protein can be capable of preventing or ameliorating disease progression in a number of diseases, including, but not limited to, AD, FTD, autism, traumatic brain injury, Parkinson’s disease, and Dravet syndrome.
  • the engineered guide RNAs of the present disclosure can target MAPT for RNA editing, thereby, driving a reduction in Tau protein expression.
  • Tau protein expression is reduced in human neurons.
  • the present disclosure provides compositions of engineered guide RNAs that target MAPT and facilitated ADAR- mediated RNA editing of MAPT to reduce pathogenic levels of Tau by targeting key adenosines for deamination that are present in the translational initiation sites (TISs).
  • the engineered guide RNAs of the present disclosure target a coding sequence in MAPT.
  • the coding sequence can be a translation initiation site (TIS) (AUG) of MAPT and the engineered guide RNA can facilitate ADAR-mediated RNA editing of AUG to GUG.
  • Engineered guide RNAs of the present disclosure can target one or more of the TISs in MAPT to reduce or completely inhibit Tau protein expression.
  • an engineered guide RNA targets the AUG at the 18 th nucleotide in Exon 1 (c.l, Nm_005910.5; GRCh37/Hgl9; also referred to as “c.1” for coding nucleotide 1), which is referred to as the conventional or canonical TIS and is also referred to as the c.1 TIS or the AUG at the 18 th nucleotide in Exon 1 corresponding to the canonical TIS at nucleotide position 151 of the MAPT reference transcript of NM_001377265.1.
  • the engineered polynucleotides of the present disclosure encode guide RNAs target the c.1 TIS and have at least 80%, 85%,
  • an engineered guide RNA targets the AUG at the 48th nucleotide in Exon 1 (c.31). In some embodiments, an engineered guide RNA targets the AUG at the 6th nucleotide in Exon 5 (c.379).
  • these three TISs correspond to methionines (Met) 1, 11 and 127, respectively.
  • an engineered guide RNA targets the AUG at the 108th nucleotide in Exon 1 (c.91).
  • one or more than one engineered guide RNAs of the present disclosure target any one or any combination of said four TISs.
  • a single engineered guide RNA of the present disclosure can be designed to target more than one of the above four TISs.
  • more than one engineered guide RNAs are designed to each independently target more than one of the above four TISs.
  • engineered guide RNAs of the present disclosure can target any one or any combination of the TISs in Exon 1 (c.l, c.31, and c.91).
  • the ratio of 3R to 4R isoforms of Tau can be measured by protein analysis (e g., using an ELISA or flow cytometry) to evaluate the effect of RNA editing, with a 1 to 1 ratio representing the ratio in healthy adult brain.
  • any of the engineered guide RNAs disclosed herein are packaged in an AAV vector and are virally delivered.
  • editing of a target sequence of a MAPT RNA by an engineered guide RNA via ADAR can be used to reduce expression of tau protein.
  • a reduction in tau can be utilized to treat a disease of condition associated with tau aggregation.
  • the disease or condition is a tauopathy.
  • Editing of a target MAPT RNA as described herein, with concomitant reduction in tau protein levels, can be utilized to reduce or prevent aggregation of tau protein.
  • one or more symptoms associated with aggregation of tau e.g. tauopathies
  • administering can be used to treat a disease or condition associated with tau pathophysiology, including treatment one of one or more symptoms associated with the disease or condition.
  • the disease or condition can be Alzheimer’s disease (AD).
  • the disease or condition can be frontotemporal dementia (FTD).
  • the disease or condition can be Parkinson’s disease.
  • the disease or condition can be progressive supranuclear palsy (PSP).
  • the disease or condition can be corticobasal degeneration (CBD).
  • the disease or condition can be chronic traumatic encephalopathy.
  • the disease or condition can be autism. In some embodiments, the disease or condition can be traumatic brain injury. In some embodiments, the disease or condition can be Dravet syndrome. In some embodiments, one or more symptoms of a disease or condition described herein can be treated by administration of an engineered guide RNA targeting MAPT RNA as described herein. For example, administration of an engineered guide RNA can be sufficient to reduce resting tremors, muscle stiffness, difficulty standing, difficulty walking, difficulty with bodily movements, involuntary movements, muscle rigidity, problems with coordination, rhythmic muscle contractions, slow bodily movement, bradykinesia, slow shuffling gait, or any combination thereof.
  • treatment of a disease or condition described herein can include improvement in cognitive manifestations associated with the disease or condition.
  • treatment can include a reduction in memory loss, a reduction in confusion, an increase in learning, an increase in communication ability, an increased attention span, an improved quality of life, increased recognition of people or objects, decrease in hallucinations, decrease in delusions, decrease in paranoia, decrease in impulsive behavior, decrease in restlessness, decrease in anxiety, decrease in repetitive statements or movement, or any combination thereof.
  • behavioral or cognitive tests can be administered to monitor the progression of treatment as a function of time.
  • administration of an engineered guide RNA of the present disclosure can be used to reduce tau protein levels through knockdown in order to treat a disease or condition associated with aberrant levels of tau (including disease associated with tau aggregation). While a reduction is obtained through administration, residual tau protein can still be present after the administering. In some cases, the presence of reduced tau protein levels treats the disease or condition, without reducing the level of tau protein levels to zero. Such levels can be determined in an in vitro assay using a sample obtained from a subject. In some cases, reduction in tau levels by administration of an engineered guide RNA of the current disclosure can manifest with improved pathophysiology associated with functional tau protein. For example, administration of an engineered guide RNA of the present disclosure can reduce neurofibrillary tangles, neuronal cell death, neuroinflammation, or other manifestations, relative to an amount prior to the administering.
  • the biomarkers can be tau isoforms.
  • various isoforms of tau protein e g. 1R, 2R, 3R, 4R 5R or 6R
  • the 4R isoform of tau can be increase or maintained upon administration.
  • the 3R isoform of tau can be decreased or maintained upon administration.
  • the ratio of the 4R isoform of tau to the 3R isoform of tau can be increase or maintained upon administration.
  • a biomarker can be post-translationally modified tau.
  • levels of post-translationally modified tau can be modulated upon administration of an engineered guide RNA of the present disclosure.
  • the level of phosphorylated tau can be decreased or maintained upon administration.
  • the ratio of phosphorylated tau to non-phosphorylated tau can be decreased or maintained upon administration.
  • biomarker levels can be determined in an in vitro assay. In some instances, biomarker levels can be determined in vivo using, for example, using an imaging technique such as MRI.
  • the engineered guide RNAs target a non-coding sequence in MAPT.
  • the non-coding sequence can be a polyA signal sequence and the engineered guide RNA can facilitate ADAR-mediated RNA editing of one or more adenosines in the polyA signal sequence of MAPT.
  • engineered guide RNAs of the present disclosure can be multiplexed to target more than one polyA signal sequences in MAPT.
  • engineered guide RNAs of the present disclosure can be multiplexed to target the TIS and one or more polyA signal sequences in MAPT.
  • engineered guide RNAs of the present disclosure targeting the canonical TIS (the c.l TIS) of MAPT can be multiplexed with one or more additional engineered guide RNAs targeting a different TIS of MAPT, such as the c.11 TIS, the c.31 TIS, or the c.91 TIS.
  • engineered guide RNAs of the present disclosure targeting the MAPT TIS can be multiplexed with one or more engineered guide RNAs of a different sequence targeting a different region of MAPT, such as the 5’UTR region of MAPT (e.g., a Kozak sequence, an internal ribosomal entry site (IRES), or an iron response element (IRE) of the 5’ UTR).
  • engineered guide RNAs can be multiplexed to target a non-coding sequence and a coding sequence in MAPT.
  • the multiplexed engineered guide RNAs can be delivered together in the same viral vector or the each of the distinct engineered guide RNAs can be delivered together but in separate vectors.
  • the engineered guide RNAs of the present disclosure facilitated ADAR-mediated RNA editing of MAPT, thereby, effecting protein knockdown.
  • an engineered guide RNA of the present disclosure can comprise multiple targeting domains that hybridize to multiple sequences within a MAPT RNA.
  • an engineered guide RNA can have a first targeting domain that hybridizes to a sequence comprising the AUG in the 18th nucleotide in Exon 1 (c.1, canonical TIS of MAPT), and a second targeting domain that hybridizes to a second sequence of the target MAPT RNA selected from the group consisting of: (a) an AUG in the 48th nucleotide in Exon 1 (c.31), (b) an AUG in the 108th nt of Exon 1 (c.91), (c) an AUG in the 6th nucleotide in Exon 5 (c.379), and any combination thereof.
  • the present disclosure provides engineered guide RNAs that facilitate edits at multiple adenosines. Hydrolytic deamination of multiple adenosines in an RNA can be referred to as hyper-editing.
  • hyper-editing can occur in cis (e.g. in an Alu element) or in trans (e.g. in a target RNA by an engineered guide RNA).
  • hyper editing can comprise editing in the polyA signal sequence of the MAPT target RNA.
  • hyper-editing can introduce edits in at least 2 or more nucleotides of a subject target RNA. In some cases, hyper-editing can introduce at least or at most about 2, 4, 6, 8, 10, 12, 14, 16, 18,
  • hyper-editing can occur in an untranslated region, translated region, 3’UTR, 5’UTR, or any combinations thereof.
  • the engineered guide RNAs of the present disclosure facilitated ADAR-mediated RNA editing of from 1 to 100% of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate from 40 to 90% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 5% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 10% editing of a target adenosine. 15% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 20% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 25% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 30% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 35% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 40% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 45% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 50% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 55% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 60% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 65% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 75% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 80% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 85% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 90% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 95% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate 100% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate from 5 to 20% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate from 20 to 40% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate from 40 to 60% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate from 60 to 80% editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate from 80 to 100% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate from 60 to 80% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate from 70 to 90% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% or more editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 80% or more editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate up to 90% or more editing of a target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 10% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 30% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 25% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 20% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 15% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 10% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 9% editing of an off- target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 8% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 7% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 6% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 5% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 4% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 3% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 2% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 1% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining 0% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 30% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 29% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 28% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 27% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 26% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 25% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 24% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 23% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 22% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 21% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 20% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 19% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 18% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 17% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 16% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 15% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 14% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 13% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 12% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 11% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 10% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 9% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 8% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 7% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 6% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 5% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 4% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 3% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 2% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining less than 1% editing of an off-target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% editing of a target adenosine while maintaining 0% editing of an off-target adenosine.
  • the engineered guide RNAs of the present disclosure facilitate ADAR-mediated RNA editing of MAPT, which results in knockdown of protein levels.
  • the knockdown in protein levels is quantitated as a reduction in expression of the Tau protein.
  • the engineered guide RNAs of the present disclosure can facilitate from 1% to 100% Tau protein knockdown.
  • the engineered guide RNAs of the present disclosure can facilitate from 1% to 10%, from 10% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to 90%, from 90% to 100%, from 20% to 40%, from 30% to 50%, from 40% to 60%, from 50% to 70%, from 60% to 80%, from 20% to 50%, from 30% to 60%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% Tau protein knockdown.
  • the engineered guide RNAs of the present disclosure facilitate from 30% to 60% Tau protein knockdown.
  • Tau protein knockdown can be measured by an assay comparing a sample or subject treated with the engineered guide RNA to a control sample or subject not treated with the engineered guide RNA.
  • An engineered guide RNA of the present disclosure can be used in a method of treating a disorder in a subject in need thereof.
  • a disorder can be a disease, a condition, a genotype, a phenotype, or any state associated with an adverse effect.
  • treating a disorder can comprise preventing, slowing progression of, reversing, or alleviating symptoms of the disorder.
  • a method of treating a disorder can comprise delivering an engineered polynucleotide encoding an engineered guide RNA to a cell of a subject in need thereof and expressing the engineered guide RNA in the cell.
  • an engineered guide RNA of the present disclosure can be used to treat a genetic disorder (e.g., a Tauopathy such as AD, FTD, Parkinson’s disease). In some embodiments, an engineered guide RNA of the present disclosure can be used to treat a condition associated with one or more mutations.
  • a genetic disorder e.g., a Tauopathy such as AD, FTD, Parkinson’s disease.
  • an engineered guide RNA of the present disclosure can be used to treat a condition associated with one or more mutations.
  • compositions described herein can be formulated with a pharmaceutically acceptable carrier for administration to a subject (e.g., a human or a non-human animal).
  • a subject e.g., a human or a non-human animal.
  • a pharmaceutically acceptable carrier can include, but is not limited to, phosphate buffered saline solution, water, emulsions (e.g., an oil/water emulsion or a water/oil emulsions), glycerol, liquid polyethylene glycols, aprotic solvents such (e.g., dimethylsulfoxide, N-methylpyrrolidone, or mixtures thereof), and various types of wetting agents, solubilizing agents, anti-oxidants, bulking agents, protein carriers such as albumins, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like.
  • phosphate buffered saline solution water
  • emulsions e.g., an oil/water emulsion or a water/oil emulsions
  • glycerol liquid polyethylene glyco
  • compositions also can include stabilizers and preservatives. Additional examples of carriers, stabilizers and adjuvants consistent with the compositions of the present disclosure can be found in, for example, Remington's Pharmaceutical Sciences, 21st Ed., Mack Publ. Co., Easton, Pa. (2005), incorporated herein by reference in its entirety.
  • the pharmaceutical composition can be formulated in unit dose forms or multiple-dose forms.
  • the unit dose forms can be physically discrete units suitable for administration to human or non-human subjects (e.g., animals).
  • the unit dose forms can be packaged individually.
  • each unit dose contains a predetermined quantity of an active ingredient(s) that can be sufficient to produce the desired therapeutic effect in association with pharmaceutical carriers, diluents, excipients, or any combination thereof.
  • the unit dose forms comprise ampules, syringes, or individually packaged tablets and capsules, or any combination thereof.
  • a unit dose form can be comprised in a disposable syringe.
  • unit-dosage forms can be administered in fractions or multiples thereof.
  • a multiple-dose form comprises a plurality of identical unit dose forms packaged in a single container, which can be administered in segregated a unit dose form.
  • multiple dose forms comprise vials, bottles of tablets or capsules, or bottles of pints or gallons.
  • a multiple- dose forms comprise the same pharmaceutically active agents.
  • a multiple- dose forms comprise different pharmaceutically active agents.
  • the pharmaceutical composition comprises a pharmaceutically acceptable excipient.
  • the excipient comprises a buffering agent, a cryopreservative, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, or a coloring agent, or any combination thereof.
  • an excipient comprises a buffering agent.
  • the buffering agent comprises sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, calcium bicarbonate, or any combination thereof.
  • the buffering agent comprises sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, or calcium hydroxide and other calcium salts, or any combination thereof.
  • an excipient comprises a cryopreservative.
  • the cryopreservative comprises DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof.
  • a cryopreservative comprises a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof.
  • an excipient comprises a pH agent (to minimize oxidation or degradation of a component of the composition), a stabilizing agent (to prevent modification or degradation of a component of the composition), a buffering agent (to enhance temperature stability), a solubilizing agent (to increase protein solubility), or any combination thereof.
  • an excipient comprises a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a triglyceride, an alcohol, or any combination thereof.
  • an excipient comprises sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HC1, disodium edetate, lecithin, glycerin, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof.
  • the excipient can be an excipient described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).
  • the excipient comprises a preservative.
  • the preservative comprises an antioxidant, such as alpha-tocopherol and ascorbate, an antimicrobial, such as parabens, chlorobutanol, and phenol, or any combination thereof.
  • the antioxidant comprises EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), sodium sulfite, p-amino benzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol or N- acetyl cysteine, or any combination thereof.
  • the preservative comprises validamycin A, TL-3, sodium ortho vanadate, sodium fluoride, N-a-tosyl-Phe- chloromethylketone, N-a-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitor, phosphatase inhibitor, caspase inhibitor, granzyme inhibitor, cell adhesion inhibitor, cell division inhibitor, cell cycle inhibitor, lipid signaling inhibitor, protease inhibitor, reducing agent, alkylating agent, antimicrobial agent, oxidase inhibitor, or other inhibitors, or any combination thereof.
  • the excipient comprises a binder.
  • the binder comprises starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, or any combination thereof.
  • the binder can be a starch, for example a potato starch, corn starch, or wheat starch; a sugar such as sucrose, glucose, dextrose, lactose, or maltodextrin; a natural and/or synthetic gum; a gelatin; a cellulose derivative such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, or ethyl cellulose; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); a wax; calcium carbonate; calcium phosphate; an alcohol such as sorbitol, xylitol, mannitol, or water, or any combination thereof.
  • a starch for example a potato starch, corn starch, or wheat starch
  • a sugar such as sucrose, glucose, dextrose, lactose, or maltodextrin
  • the excipient comprises a lubricant.
  • the lubricant comprises magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, or light mineral oil, or any combination thereof.
  • the lubricant comprises metallic stearates (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffins, hydrogenated vegetable oils, leucine, polyethylene glycols (PEG), metallic lauryl sulphates (such as sodium lauryl sulphate, magnesium lauryl sulphate), sodium chloride, sodium benzoate, sodium acetate or talc or a combination thereof.
  • metallic stearates such as magnesium stearate, calcium stearate, aluminum stearate
  • fatty acid esters such as sodium stearyl fumarate
  • fatty acids such as stearic acid
  • fatty alcohols such as sodium stearic acid
  • fatty alcohols such as sodium stearyl fumarate
  • fatty acids such as stearic acid
  • the excipient comprises a dispersion enhancer.
  • the dispersion enhancer comprises starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isomorphous silicate, or microcrystalline cellulose, or any combination thereof as high HLB emulsifier surfactants.
  • the excipient comprises a disintegrant.
  • a disintegrant comprises a non-effervescent disintegrant.
  • a non-effervescent disintegrants comprises starches such as com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, or gums such as agar, guar, locust bean, karaya, pectin, and tragacanth, or any combination thereof.
  • a disintegrant comprises an effervescent disintegrant.
  • a suitable effervescent disintegrant comprises bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • the excipient comprises a sweetener, a flavoring agent or both.
  • a sweetener comprises glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as a sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like, or any combination thereof.
  • flavoring agents incorporated into a composition comprise synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; or any combination thereof.
  • a flavoring agent comprises a cinnamon oils; oil of wintergreen; peppermint oils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oil such as lemon oil, orange oil, grape and grapefruit oil, and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot, or any combination thereof.
  • the excipient comprises a pH agent (e.g., to minimize oxidation or degradation of a component of the composition), a stabilizing agent (e g., to prevent modification or degradation of a component of the composition), a buffering agent (e.g., to enhance temperature stability), a solubilizing agent (e.g., to increase protein solubility), or any combination thereof.
  • the excipient comprises a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a trigylceride, an alcohol, or any combination thereof.
  • the excipient comprises sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HC1, disodium edetate, lecithin, glycerine, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof.
  • PEG poly-ethylene glycol
  • HSA human serum albumin
  • the excipient comprises a cryo-preservative.
  • the excipient comprises DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof.
  • the excipient comprises a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof.
  • the pharmaceutical composition comprises a diluent.
  • the diluent comprises water, glycerol, methanol, ethanol, or other similar biocompatible diluents, or any combination thereof.
  • a diluent comprises an aqueous acid such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or any combination thereof.
  • a diluent comprises an alkaline metal carbonates such as calcium carbonate; alkaline metal phosphates such as calcium phosphate; alkaline metal sulphates such as calcium sulphate; cellulose derivatives such as cellulose, microcrystalline cellulose, cellulose acetate; magnesium oxide, dextrin, fructose, dextrose, glyceryl palmitostearate, lactitol, choline, lactose, maltose, mannitol, simethicone, sorbitol, starch, pregelatinized starch, talc, xylitol and/or anhydrates, hydrates and/or pharmaceutically acceptable derivatives thereof or combinations thereof.
  • alkaline metal carbonates such as calcium carbonate
  • alkaline metal phosphates such as calcium phosphate
  • alkaline metal sulphates such as calcium sulphate
  • cellulose derivatives such as cellulose, microcrystalline cellulose, cellulose acetate
  • magnesium oxide de
  • the pharmaceutical composition comprises a carrier.
  • the carrier comprises a liquid or solid filler, solvent, or encapsulating material.
  • the carrier comprises additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldolic acids, esterified sugars and the like; and polysaccharides or sugar polymers), alone or in combination.
  • An engineered guide RNA of the present disclosure (such as an engineered guide RNA with a polynucleotide sequence of any one of SEQ ID NO: 2-1215 as recited in Table 1 that target the MAPT c.l TIS) or an engineered polynucleotide of the present disclosure (e.g., an engineered polynucleotide encoding an engineered guide RNA) can be delivered via a delivery vehicle.
  • the delivery vehicle is a vector.
  • a vector can facilitate delivery of the engineered polynucleotide into a cell to genetically modify the cell.
  • the vector comprises DNA, such as double stranded or single stranded DNA.
  • the delivery vector can be a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector or plasmid), a viral vector, or any combination thereof.
  • the vector is an expression cassette.
  • a viral vector comprises a viral capsid, an inverted terminal repeat sequence, and the engineered polynucleotide can be used to deliver the engineered guide RNA to a cell.
  • the viral vector can be a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, an alphavirus vector, a lentivirus vector (e.g., human or porcine), a Herpes virus vector, an Epstein-Barr virus vector, an SV40 virus vectors, a pox virus vector, or a combination thereof.
  • the viral vector can be a recombinant vector, a hybrid vector, a chimeric vector, a self-complementary vector, a single-stranded vector, or any combination thereof.
  • the viral vector can be an adeno-associated virus (AAV).
  • AAV can be any AAV known in the art.
  • the viral vector can be of a specific serotype.
  • the viral vector can be an AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV10 serotype, AAV11 serotype, AAV 12 serotype, AAV13 serotype, AAV 14 serotype, AAV 15 serotype, AAV16 serotype, AAV.rh8 serotype, AAV.rhlO serotype, AAV.rh20 serotype, AAV.rh39 serotype, AAV.Rh74 serotype, AAV.RHM4-1 serotype, AAV.hu37 sero
  • the AAV vector can be a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV, or any combination thereof.
  • scAAV self-complementary AAV
  • the AAV vector can be a recombinant AAV (rAAV) vector.
  • rAAV recombinant AAV
  • Methods of producing recombinant AAV vectors can be known in the art and generally involve, in some cases, introducing into a producer cell line: (1) DNA necessary for AAV replication and synthesis of an AAV capsid, (b) one or more helper constructs comprising the viral functions missing from the AAV vector, (c) a helper virus, and (d) the plasmid construct containing the genome of the AAV vector, e.g., ITRs, promoter and engineered guide RNA sequences, etc.
  • the viral vectors described herein can be engineered through synthetic or other suitable means by references to published sequences, such as those that can be available in the literature.
  • published sequences such as those that can be available in the literature.
  • the genomic and protein sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits can be known in the art and can be found in the literature or in public databases such as GenBank or Protein Data Bank (PDB).
  • methods of producing delivery vectors herein comprising packaging an engineered polynucleotide of the present disclosure (e.g., an engineered polynucleotide encoding an engineered guide RNA) in an AAV vector.
  • an engineered polynucleotide of the present disclosure e.g., an engineered polynucleotide encoding an engineered guide RNA
  • methods of producing the delivery vectors described herein comprise, (a) introducing into a cell: (i) a polynucleotide comprising a promoter and an engineered guide RNA disclosed herein; and (ii) a viral genome comprising a Replication (Rep) gene and Capsid (Cap) gene that encodes a wild- type AAV capsid protein or modified version thereof; (b) expressing in the cell the wild-type AAV capsid protein or modified version thereof; (c) assembling an AAV particle; and (d) packaging the engineered guide RNA disclosed herein in the AAV particle, thereby generating an AAV delivery vector.
  • the recombinant vectors comprise one or more inverted terminal repeats and the inverted terminal repeats comprise a 5’ inverted terminal repeat, a 3’ inverted terminal repeat, and a mutated inverted terminal repeat.
  • the mutated terminal repeat lacks a terminal resolution site, thereby enabling formation of a self complementary AAV.
  • a hybrid AAV vector can be produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes can be not the same.
  • ITR inverted terminal repeat
  • the Rep gene and ITR from a first AAV serotype e.g., AAV2
  • a second AAV serotype e.g., AAV5 or AAV9
  • a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein can be indicated AAV2/9.
  • the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/6, AAV2/8, or AAV2/9 vector.
  • the AAV vector can be a chimeric AAV vector.
  • the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes.
  • a chimeric AAV vector can be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.
  • the AAV vector comprises a self-complementary AAV genome. Self-complementary AAV genomes can be generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.
  • the delivery vector can be a retroviral vector.
  • the retroviral vector can be a Moloney Murine Leukemia Virus vector, a spleen necrosis virus vector, or a vector derived from the Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, or mammary tumor virus, or a combination thereof
  • the retroviral vector can be transfected such that the majority of sequences coding for the structural genes of the virus (e.g., gag, pol, and env) can be deleted and replaced by the gene(s) of interest.
  • the delivery vehicle can be a non-viral vector.
  • the delivery vehicle can be a plasmid.
  • the plasmid comprises DNA.
  • the plasmid comprises circular double-stranded DNA.
  • the plasmid can be linear.
  • the plasmid comprises one or more genes of interest and one or more regulatory elements.
  • the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria.
  • the plasmid can be a minicircle plasmid.
  • the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid.
  • the plasmid can be formulated for delivery through injection by a needle carrying syringe.
  • the plasmid can be formulated for delivery via electroporation.
  • the plasmids can be engineered through synthetic or other suitable means known in the art.
  • the genetic elements can be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which can then be readily ligated to another genetic sequence.
  • the vector containing the engineered guide RNA or the engineered polynucleotide is a non-viral vector system.
  • the non-viral vector system comprises cationic lipids, or polymers.
  • the non-viral vector system comprises can be a liposome or polymeric nanoparticle.
  • the engineered guide RNA or a non-viral vector comprising the engineered guide RNA is delivered to a cell by hydrodynamic injection or ultrasound.
  • Administration can refer to methods that can be used to enable the delivery of a composition described herein (e.g. comprising an engineered guide RNA or an engineered polynucleotide encoding the same) to the desired site of biological action.
  • a composition described herein e.g. comprising an engineered guide RNA or an engineered polynucleotide encoding the same
  • an engineered guide RNA such as an engineered guide RNA with a polynucleotide sequence of any one of SEQ ID NO: 2-1215 as recited in Table 1 that targets the MAPT c.l TIS
  • Administration disclosed herein to an area in need of treatment or therapy can be achieved by, for example, and not by way of limitation, oral administration, topical administration, intravenous administration, inhalation administration, or any combination thereof.
  • delivery can include inhalation, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebroventricular, intracistemal, intracorneal, intracoronal, intracoronary, intracorpous cavernaosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal
  • Delivery can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion), oral administration, inhalation administration, intraduodenal administration, rectal administration, or a combination thereof. Delivery can include direct application to the affected tissue or region of the body.
  • topical administration can comprise administering a lotion, a solution, an emulsion, a cream, a balm, an oil, a paste, a stick, an aerosol, a foam, a jelly, a foam, a mask, a pad, a powder, a solid, a tincture, a butter, a patch, a gel, a spray, a drip, a liquid formulation, an ointment to an external surface of a surface, such as a skin.
  • Delivery can include a parenchymal injection, an intra-thecal injection, an intra-ventricular injection, or an intra-ci sternal injection.
  • a composition provided herein can be administered by any method.
  • a method of administration can be by intra-arterial injection, intracistemal injection, intramuscular injection, intraparenchymal injection, intraperitoneal injection, intraspinal injection, intrathecal injection, intravenous injection, intraventricular injection, stereotactic injection, subcutaneous injection, epidural, or any combination thereof.
  • Delivery can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion administration).
  • delivery can comprise a nanoparticle, a liposome, an exosome, an extracellular vesicle, an implant, or a combination thereof.
  • delivery can be from a device.
  • delivery can be administered by a pump, an infusion pump, or a combination thereof. In some embodiments, delivery can be by an enema, an eye drop, a nasal spray, or any combination thereof. In some instances, a subject can administer the composition in the absence of supervision. In some instances, a subject can administer the composition under the supervision of a medical professional (e g., a physician, nurse, physician’s assistant, orderly, hospice worker, etc.). In some embodiments, a medical professional can administer the composition.
  • a medical professional e g., a physician, nurse, physician’s assistant, orderly, hospice worker, etc.
  • administering can be oral ingestion.
  • delivery can be a capsule or a tablet.
  • Oral ingestion delivery can comprise a tea, an elixir, a food, a drink, a beverage, a syrup, a liquid, a gel, a capsule, a tablet, an oil, a tincture, or any combination thereof.
  • a food can be a medical food.
  • a capsule can comprise hydroxymethylcellulose.
  • a capsule can comprise a gelatin, hydroxypropylmethyl cellulose, pullulan, or any combination thereof
  • capsules can comprise a coating, for example, an enteric coating.
  • a capsule can comprise a vegetarian product or a vegan product such as a hypromellose capsule.
  • delivery can comprise inhalation by an inhaler, a diffuser, a nebulizer, a vaporizer, or a combination thereof.
  • disclosed herein can be a method, comprising administering a composition disclosed herein to a subject (e g., a human) in need thereof.
  • a subject e g., a human
  • the method can treat (including prevent) a disease in the subject.
  • a pharmaceutical composition disclosed herein can be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, or prophylactic, effect.
  • the appropriate dosage and treatment regimen for the methods of treatment described herein vary with respect to the particular disease being treated, the gRNA and/or ADAR (or a vector encoding the gRNA and/or ADAR) being delivered, and the specific condition of the subject.
  • the administration can be over a period of time until the desired effect (e.g., reduction in symptoms can be achieved).
  • administration can be 1, 2, 3, 4, 5, 6, or 7 times per week.
  • administration or application of a composition disclosed herein can be performed for a treatment duration of at least about 1 week, at least about 1 month, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more.
  • administration can be over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
  • administration can be over a period of 2, 3, 4, 5, 6 or more months.
  • administration can be performed repeatedly over a lifetime of a subject, such as once a month or once a year for the lifetime of a subject. In some examples, administration can be performed repeatedly over a substantial portion of a subject’s life, such as once a month or once a year for at least about 1 year, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, or more. In some examples, treatment can be resumed following a period of remission.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a “bulge” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand.
  • a bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold.
  • a bulge does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair - a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a “mismatch.” Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an “internal loop.”
  • a “symmetrical bulge” refers to a bulge where the same number of nucleotides is present on each side of the bulge. An “asymmetrical bulge” refers to a bulge where a different number of nucleotides are present on each side of the bulge.
  • complementary refers to the ability of a nucleic acid to form one or more bonds with a corresponding nucleic acid sequence by, for example, hydrogen bonding (e g., traditional Watson-Crick), covalent bonding, or other similar methods.
  • a double hydrogen bond forms between nucleobases T and A, whereas a triple hydrogen bond forms between nucleobases C and G.
  • the sequence A-G-T can be complementary to the sequence T-C-A.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson- Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).
  • Perfectly complementary can mean that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein can refer to a degree of complementarity that can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides, or can refer to two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions). Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” or “not specific” can refer to a nucleic acid sequence that contains a series of residues that can be not designed to be complementary to or can be only partially complementary to any other nucleic acid sequence.
  • determining can be used interchangeably herein to refer to forms of measurement.
  • the terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • encode refers to an ability of a polynucleotide to provide information or instructions sequence sufficient to produce a corresponding gene expression product.
  • mRNA can encode a polypeptide during translation
  • DNA can encode an mRNA molecule during transcription.
  • An “engineered latent guide RNA” refers to an engineered guide RNA that comprises a portion of sequence that, upon hybridization or only upon hybridization to a target RNA, substantially forms at least a portion of a structural feature, other than a single A/C mismatch feature at the target adenosine to be edited.
  • the term “facilitates RNA editing” by an engineered guide RNA refers to the ability of the engineered guide RNA when associated with an RNA editing entity and a target RNA to provide a targeted edit of the target RNA by the RNA edited entity.
  • the engineered guide RNA can directly recruit or position/orient the RNA editing entity to the proper location for editing of the target RNA.
  • the engineered guide RNA when hybridized to the target RNA forms a guide-target RNA scaffold with one or more structural features as described herein, where the guide-target RNA scaffold with structural features recruits or positions/orients the RNA editing entity to the proper location for editing of the target RNA.
  • a “guide-target RNA scaffold,” as disclosed herein, is the resulting double stranded RNA formed upon hybridization of a guide RNA, with latent structure, to a target RNA.
  • a guide-target RNA scaffold has one or more structural features formed within the double stranded RNA duplex upon hybridization.
  • the guide-target RNA scaffold can have one or more structural features selected from a bulge, mismatch, internal loop, hairpin, or wobble base pair.
  • a “hairpin” includes an RNA duplex wherein a portion of a single RNA strand has folded in upon itself to form the RNA duplex.
  • the portion of the single RNA strand folds upon itself due to having nucleotide sequences that base pair to each other, where the nucleotide sequences are separated by an intervening sequence that does not base pair with itself, thus forming a base-paired portion and non-base paired, intervening loop portion.
  • the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, can refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
  • the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters
  • percent identity and sequence similarity can be performed using the BLAST algorithm, which is described in Altschul et al. (L Mol. Biol. 215:403-410 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • an “internal loop” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, has 5 nucleotides or more. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a “bulge” or a “mismatch,” depending on the size of the structural feature.
  • a “symmetrical internal loop” is formed when the same number of nucleotides is present on each side of the internal loop. An “asymmetrical internal loop” is formed when a different number of nucleotides is present on each side of the internal loop.
  • “Latent structure” refers to a structural feature that substantially forms only upon hybridization of a guide RNA to a target RNA.
  • the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA.
  • the structural feature is formed and the latent structure provided in the guide RNA is, thus, unmasked.
  • RNA molecules comprising a sequence that encodes a polypeptide or protein.
  • RNA can be transcribed from DNA.
  • precursor mRNA containing non-protein coding regions in the sequence can be transcribed from DNA and then processed to remove all or a portion of the non-coding regions (introns) to produce mature mRNA.
  • pre-mRNA can refer to the RNA molecule transcribed from DNA before undergoing processing to remove the non-protein coding regions.
  • a mismatch refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNA scaffold.
  • a mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceeds 1, the resulting structure is no longer considered a mismatch, but rather, is considered a “bulge” or an “internal loop,” depending on the size of the structural feature.
  • polynucleotide can refer to a single or double- stranded polymer of deoxyribonucleotide (DNA) or ribonucleotide (RNA) bases read from the 5’ to the 3’ end.
  • DNA deoxyribonucleotide
  • RNA ribonucleotide
  • RNA is inclusive of dsRNA (double stranded RNA), snRNA (small nuclear RNA), IncRNA (long non-coding RNA), mRNA (messenger RNA), miRNA (microRNA) RNAi (inhibitory RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), snoRNA (small nucleolar RNA), and cRNA (complementary RNA).
  • DNA is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids.
  • protein can be used interchangeably and in their broadest sense can refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide can contain at least two amino acids and no limitation can be placed on the maximum number of amino acids which can comprise a protein’s or peptide's sequence.
  • amino acid can refer to either natural amino acids, unnatural amino acids, or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • fusion protein can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function.
  • linker can refer to a protein fragment that can be used to link these domains together - optionally to preserve the conformation of the fused protein domains, prevent unfavorable interactions between the fused protein domains which can compromise their respective functions, or both.
  • structured motif refers to a combination of two or more structural features in a guide-target RNA scaffold.
  • a “subject” refers to a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be a mammal.
  • the mammal can be a human.
  • the subject can be diagnosed or suspected of being at high risk for a disease. In some cases, the subject may not be necessarily diagnosed or suspected of being at high risk for the disease [00231]
  • the term “in vivo” refers to an event that takes place in a subject’s body.
  • ex vivo refers to an event that takes place outside of a subject’s body.
  • An ex vivo assay may not be performed on a subject. Rather, it can be performed upon a sample separate from a subject.
  • An example of an ex vivo assay performed on a sample can be an “in vitro” assay.
  • in vitro refers to an event that takes places contained in a container for holding laboratory reagent such that it can be separated from the biological source from which the material can be obtained.
  • in vitro assays can encompass cell-based assays in which living or dead cells can be employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells can be employed.
  • wobble base pair refers to two bases that weakly pair.
  • a wobble base pair can refer to a G paired with a U.
  • substantially forms as described herein, when referring to a particular secondary structure, refers to formation of at least 80% of the structure under physiological conditions (e.g . physiological pH, physiological temperature, physiological salt concentration, etc ).
  • treatment or “treating” can be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results include but can be not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit can refer to eradication or amelioration of one or more symptoms of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement can be observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of one or more symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease may not have been made.
  • compositions, and methods are disclosed herein. Specific exemplary embodiments of these compositions and methods are disclosed below. The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed.
  • Embodiment 1 A composition comprising an engineered guide RNA, wherein: a) the engineered guide RNA, upon hybridization to a sequence of a target MAPT RNA, forms a guide- target RNA scaffold with the sequence of the target MAPT RNA; b) formation of the guide- target RNA scaffold substantially forms one or more structural features selected from the group consisting of: a bulge, an internal loop, and a hairpin; and c) the structural feature is not present within the engineered guide RNA prior to the hybridization of the engineered guide RNA to the MAPT target RNA; and d) upon hybridization of the engineered guide RNA to the sequence of the target MAPT RNA, the engineered guide RNA facilitates RNA editing of one or more target adenosines in the sequence of the target MAPT RNA by an RNA editing entity.
  • Embodiment 2 The composition of Embodiment 1, wherein the sequence of the target MAPT RNA is within the 3’ untranslated region (UTR).
  • Embodiment 3. The composition of Embodiment 1, wherein the sequence of the target MAPT RNA is within the 5’ untranslated region (UTR).
  • Embodiment 4. The composition of Embodiment 3, wherein the sequence of the target MAPT RNA in the 5’ UTR is a Kozak sequence.
  • the composition of Embodiment 3, wherein the sequence of the target MAPT RNA in the 5’ UTR is an internal ribosomal entry site (IRES).
  • Embodiment 3 wherein the sequence of the target MAPT RNA in the 5’ UTR is an iron response element (IRE).
  • Embodiment 7 The composition of Embodiment 1, wherein the sequence of the target MAPT RNA comprises a translation initiation site (TIS).
  • Embodiment 8 The composition of Embodiment 7, wherein the translation initiation site is an AUG starting at the 18th nucleotide in Exon 1 (c.1).
  • Embodiment 9 The composition of Embodiment 7, wherein the translation initiation site is the c.l TIS corresponding to the canonical TIS at nucleotide position 151 of the MAPT reference transcript of NM_001377265.1.
  • composition of any one of Embodiments 7-9, wherein the one or more structural features comprises: a first 6/6 symmetric internal loop at a position selected from the group consisting of: 34, 33, 32, 31, 30, 26, 25, 24, 23, and 22; relative to the target adenosine at position 0.
  • Embodiment 11 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 34, relative to the target adenosine at position 0.
  • composition of Embodiment 11 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • Embodiment 13 The composition of Embodiment 12, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 478.
  • Embodiment 14 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 478.
  • Embodiment 13 wherein the engineered guide RNA comprises SEQ ID NO: 478.
  • Embodiment 15 The composition of Embodiment 11, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • Embodiment 16 Embodiment 16.
  • Embodiment 15 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 479.
  • Embodiment 17 The composition of Embodiment 16, wherein the engineered guide RNA comprises SEQ ID NO: 479.
  • Embodiment 18 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 33, relative to the target adenosine at position 0.
  • Embodiment 19 The composition of Embodiment 15, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 479.
  • Embodiment 17 The composition of Embodiment 16, wherein the engineered guide RNA comprises SEQ ID NO: 479.
  • Embodiment 18 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 33, relative to the target adenos
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • composition of Embodiment 19 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 592.
  • Embodiment 21 The composition of Embodiment 20, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 22 The composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 23 Embodiment 23.
  • composition of Embodiment 22, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 593.
  • the composition of Embodiment 23, wherein the engineered guide RNA comprises SEQ ID NO: 593.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/U mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 26 Embodiment 26.
  • Embodiment 25 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 639.
  • Embodiment 27 The composition of Embodiment 26, wherein the engineered guide RNA comprises SEQ ID NO: 639.
  • Embodiment 28 The composition of Embodiment 25, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 639.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/U mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 29 Embodiment 29.
  • Embodiment 28 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 640.
  • Embodiment 30 The composition of Embodiment 29, wherein the engineered guide RNA comprises SEQ ID NO: 640.
  • Embodiment 31 The composition of Embodiment 28, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 640.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, and any combination thereof.
  • Embodiment 32 The composition of Embodiment 31, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 686.
  • Embodiment 33 The composition of Embodiment 32, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 34 The composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 35 The composition of Embodiment 34, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 687.
  • Embodiment 36 The composition of Embodiment 35, wherein the engineered guide RNA comprises SEQ ID NO: 687.
  • Embodiment 37 The composition of Embodiment 35, wherein the engineered guide RNA comprises SEQ ID NO: 687.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 38 Embodiment 38.
  • Embodiment 37 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 688.
  • Embodiment 39 The composition of Embodiment 38, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 40 The composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof Embodiment 41.
  • Embodiment 40 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 695.
  • Embodiment 42 The composition of Embodiment 41, wherein the engineered guide RNA comprises SEQ ID NO: 695.
  • Embodiment 43 The composition of Embodiment 40, wherein the engineered guide RNA comprises SEQ ID NO: 695.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/U Wobble at position 6 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 44 Embodiment 44.
  • Embodiment 43 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 696.
  • Embodiment 45 The composition of Embodiment 44, wherein the engineered guide RNA comprises SEQ ID NO: 696.
  • Embodiment 46 The composition of Embodiment 46.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G U Wobble at position 6 relative to position 0, a G/G mismatch at position 8 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 47 Embodiment 47.
  • Embodiment 46 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 714.
  • Embodiment 48 The composition of Embodiment 47, wherein the engineered guide RNA comprises SEQ ID NO: 714.
  • Embodiment 49 The composition of Embodiment 46, wherein the engineered guide RNA comprises SEQ ID NO: 714.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a G/G mismatch at position 8 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 50 Embodiment 50.
  • Embodiment 49 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 715.
  • Embodiment 51 The composition of Embodiment 50, wherein the engineered guide RNA comprises SEQ ID NO: 715.
  • Embodiment 52 The composition of Embodiment 49, wherein the engineered guide RNA comprises SEQ ID NO: 715.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 53 The composition of Embodiment 52, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 722.
  • Embodiment 54 The composition of Embodiment 53, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 55 The composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 56 Embodiment 56.
  • Embodiment 55 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 723.
  • Embodiment 57 The composition of Embodiment 56, wherein the engineered guide RNA comprises SEQ ID NO: 723.
  • Embodiment 58 The composition of Embodiment 58.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 3/3 symmetric bulge at position -13 relative to position 0, a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 59 The composition of Embodiment 58, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1059.
  • Embodiment 60 The composition of Embodiment 59, wherein the engineered guide RNA comprises SEQ ID NO: 1059.
  • the composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, an A/C mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 62 The composition of Embodiment 61, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1116.
  • Embodiment 63 The composition of Embodiment 62, wherein the engineered guide RNA comprises SEQ ID NO: 1116.
  • Embodiment 64 The composition of Embodiment 64.
  • composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, an A C mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 65 Embodiment 65.
  • Embodiment 64 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1117.
  • Embodiment 66 The composition of Embodiment 65, wherein the engineered guide RNA comprises SEQ ID NO: 1117.
  • Embodiment 67 The composition of Embodiment 18, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 68 The composition of Embodiment 67, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1215.
  • Embodiment 69 The composition of Embodiment 68, wherein the engineered guide RNA comprises SEQ ID NO: 1215.
  • Embodiment 70 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 32, relative to the target adenosine at position 0.
  • Embodiment 71 Embodiment 71.
  • Embodiment 70 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • Embodiment 72 The composition of Embodiment 71, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 438.
  • Embodiment 73 The composition of Embodiment 72, wherein the engineered guide RNA comprises SEQ ID NO: 438.
  • Embodiment 74. The composition of Embodiment 70, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • Embodiment 74 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 439.
  • Embodiment 76 The composition of Embodiment 75, wherein the engineered guide RNA comprises SEQ ID NO: 439.
  • Embodiment 77 The composition of Embodiment 75, wherein the engineered guide RNA comprises SEQ ID NO: 439.
  • composition of Embodiment 70 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • Embodiment 78 Embodiment 78.
  • Embodiment 77 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 816.
  • Embodiment 79 The composition of Embodiment 78, wherein the engineered guide RNA comprises SEQ ID NO: 816.
  • Embodiment 80 The composition of Embodiment 80.
  • Embodiment 70 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • Embodiment 81 The composition of Embodiment 80, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 817.
  • Embodiment 82 The composition of Embodiment 81, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 83 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 31, relative to the target adenosine at position 0.
  • Embodiment 84. The composition of Embodiment 83, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • Embodiment 84 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 162.
  • Embodiment 86 The composition of Embodiment 85, wherein the engineered guide RNA comprises SEQ ID NO: 162.
  • Embodiment 87 The composition of Embodiment 87.
  • composition of Embodiment 83 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • Embodiment 88. The composition of Embodiment 87, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 163.
  • Embodiment 89. The composition of Embodiment 88, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 90 The composition of Embodiment 83, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • Embodiment 91 Embodiment 91.
  • Embodiment 90 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 929.
  • Embodiment 92 The composition of Embodiment 91, wherein the engineered guide RNA comprises SEQ ID NO: 929.
  • Embodiment 93 The composition of Embodiment 91, wherein the engineered guide RNA comprises SEQ ID NO: 929.
  • composition of Embodiment 83 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • Embodiment 94 The composition of Embodiment 93, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 930.
  • Embodiment 95 The composition of Embodiment 94, wherein the engineered guide RNA comprises SEQ ID NO: 930.
  • Embodiment 96 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 30, relative to the target adenosine at position 0.
  • Embodiment 97 The composition of Embodiment 97.
  • Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -21 relative to position 0, a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 98 The composition of Embodiment 97, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 538.
  • Embodiment 99 The composition of Embodiment 98, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 100 The composition of Embodiment 96, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -9 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 101 The composition of Embodiment 100, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 539.
  • Embodiment 102 The composition of Embodiment 101, wherein the engineered guide RNA comprises SEQ ID NO:
  • Embodiment 103 The composition of Embodiment 96, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 4 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 104 Embodiment 104.
  • Embodiment 103 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 604.
  • Embodiment 105 The composition of Embodiment 104, wherein the engineered guide RNA comprises SEQ ID NO: 604.
  • Embodiment 106 The composition of Embodiment 106.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 4 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 107 The composition of Embodiment 106, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 605.
  • Embodiment 108 The composition of Embodiment 107, wherein the engineered guide RNA comprises SEQ ID NO: 605.
  • Embodiment 109. The composition of Embodiment 96, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 110 The composition of Embodiment 109, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 619.
  • Embodiment 111 The composition of Embodiment 110, wherein the engineered guide RNA comprises SEQ ID NO: 619.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 113 Embodiment 113.
  • Embodiment 112 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 620.
  • Embodiment 114 The composition of Embodiment 113, wherein the engineered guide RNA comprises SEQ ID NO: 620.
  • Embodiment 115 The composition of Embodiment 112, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 620.
  • Embodiment 114 The composition of Embodiment 113, wherein the engineered guide RNA comprises SEQ ID NO: 620.
  • Embodiment 115 Embodiment 115.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 116 Embodiment 116.
  • Embodiment 115 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 677.
  • Embodiment 117 The composition of Embodiment 116, wherein the engineered guide RNA comprises SEQ ID NO: 677.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 119 Embodiment 119.
  • Embodiment 118 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 678.
  • Embodiment 120 The composition of Embodiment 119, wherein the engineered guide RNA comprises SEQ ID NO: 678.
  • Embodiment 121 The composition of Embodiment 118, wherein the engineered guide RNA comprises SEQ ID NO: 678.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 122 Embodiment 122.
  • Embodiment 121 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 684.
  • Embodiment 123 The composition of Embodiment 122, wherein the engineered guide RNA comprises SEQ ID NO: 684.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a C/U mismatch at position 11 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 125 Embodiment 125.
  • Embodiment 124 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 685.
  • Embodiment 126 The composition of Embodiment 125, wherein the engineered guide RNA comprises SEQ ID NO: 685.
  • Embodiment 127 The composition of Embodiment 124, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 685.
  • Embodiment 126 The composition of Embodiment 125, wherein the engineered guide RNA comprises SEQ ID NO: 685.
  • Embodiment 127 Embodiment 127.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 128 Embodiment 128.
  • Embodiment 127 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 720.
  • Embodiment 129 The composition of Embodiment 128, wherein the engineered guide RNA comprises SEQ ID NO: 720.
  • Embodiment 130 The composition of Embodiment 127, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 720.
  • Embodiment 129 The composition of Embodiment 128, wherein the engineered guide RNA comprises SEQ ID NO: 720.
  • Embodiment 130 Embodiment 130.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 130 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 721.
  • Embodiment 132 The composition of Embodiment 131, wherein the engineered guide RNA comprises SEQ ID NO: 721.
  • Embodiment 133 The composition of Embodiment 130, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 721.
  • Embodiment 132 The composition of Embodiment 131, wherein the engineered guide RNA comprises SEQ ID NO: 721.
  • Embodiment 133 Embodiment 133.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/A mismatch at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 134 Embodiment 134.
  • Embodiment 133 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 733.
  • Embodiment 135. The composition of Embodiment 134, wherein the engineered guide RNA comprises SEQ ID NO: 733.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/A mismatch at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 137 Embodiment 137.
  • Embodiment 136 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 734.
  • Embodiment 138 The composition of Embodiment 137, wherein the engineered guide RNA comprises SEQ ID NO: 734.
  • Embodiment 139 The composition of Embodiment 136, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 734.
  • Embodiment 138 The composition of Embodiment 137, wherein the engineered guide RNA comprises SEQ ID NO: 734.
  • Embodiment 139 Embodiment 139.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 140 Embodiment 140.
  • Embodiment 139 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 763.
  • Embodiment 141 The composition of Embodiment 140, wherein the engineered guide RNA comprises SEQ ID NO: 763.
  • Embodiment 142 The composition of Embodiment 140, wherein the engineered guide RNA comprises SEQ ID NO: 763.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 143 Embodiment 143.
  • Embodiment 142 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 764.
  • Embodiment 144 The composition of Embodiment 143, wherein the engineered guide RNA comprises SEQ ID NO: 764.
  • Embodiment 145 The composition of Embodiment 142, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 764.
  • Embodiment 144 The composition of Embodiment 143, wherein the engineered guide RNA comprises SEQ ID NO: 764.
  • Embodiment 145 Embodiment 145.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/G mismatch at position 8 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 146 Embodiment 146.
  • Embodiment 145 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 775.
  • Embodiment 147 The composition of Embodiment 146, wherein the engineered guide RNA comprises SEQ ID NO: 775.
  • Embodiment 148 The composition of Embodiment 148.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 7/7 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 2 relative to position 0, a G/G mismatch at position 8 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 149 Embodiment 149.
  • Embodiment 148 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 776.
  • Embodiment 150 The composition of Embodiment 149, wherein the engineered guide RNA comprises SEQ ID NO: 776.
  • Embodiment 151 The composition of Embodiment 148, wherein the engineered guide RNA comprises SEQ ID NO: 776.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 152 Embodiment 152.
  • Embodiment 151 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 795.
  • Embodiment 153 The composition of Embodiment 152, wherein the engineered guide RNA comprises SEQ ID NO: 795.
  • Embodiment 154 The composition of Embodiment 151, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 795.
  • composition of Embodiment 96 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 42 relative to position 0, a 3/3 symmetric bulge at position 51 relative to position 0, a 3/3 symmetric bulge at position 60 relative to position 0, and any combination thereof.
  • Embodiment 155 Embodiment 155.
  • Embodiment 154 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 796.
  • Embodiment 156 The composition of Embodiment 155, wherein the engineered guide RNA comprises SEQ ID NO: 796.
  • Embodiment 157 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • Embodiment 158 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 26, relative to the target adenosine at position 0.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 159 Embodiment 159.
  • Embodiment 158 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 464.
  • Embodiment 160 The composition of Embodiment 159, wherein the engineered guide RNA comprises SEQ ID NO: 464.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof Embodiment 162.
  • Embodiment 163 The composition of Embodiment 162, wherein the engineered guide RNA comprises SEQ ID NO: 465.
  • Embodiment 164. The composition of Embodiment 157, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/A mismatch at position 11 relative to position 0, a G/U Wobble at position 18 relative to position 0, and any combination thereof.
  • Embodiment 164 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 583.
  • Embodiment 166 The composition of Embodiment 165, wherein the engineered guide RNA comprises SEQ ID NO: 583.
  • Embodiment 167 The composition of Embodiment 167.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/A mismatch at position 11 relative to position 0, a G/U Wobble at position 18 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 168 The composition of Embodiment 167, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 584.
  • Embodiment 169. The composition of Embodiment 167, wherein the engineered guide RNA comprises SEQ ID NO: 584.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a U/U mismatch at position -11 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 10 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 171 The composition of Embodiment 170, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 589.
  • Embodiment 172 The composition of Embodiment 171, wherein the engineered guide RNA comprises SEQ ID NO: 589.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a G/U Wobble at position 18 relative to position 0, a G/U Wobble at position 20 relative to position 0, and any combination thereof.
  • Embodiment 174 The composition of Embodiment 173, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 610.
  • Embodiment 175. The composition of Embodiment 174, wherein the engineered guide RNA comprises SEQ ID NO: 610.
  • Embodiment 176. The composition of Embodiment 157, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a G/U Wobble at position 18 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 177 The composition of Embodiment 176, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 611.
  • Embodiment 178 The composition of Embodiment 177, wherein the engineered guide RNA comprises SEQ ID NO: 611.
  • Embodiment 179 The composition of Embodiment 177, wherein the engineered guide RNA comprises SEQ ID NO: 611.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/G mismatch at position 18 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 180 Embodiment 180.
  • Embodiment 179 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 618.
  • Embodiment 181. The composition of Embodiment 180, wherein the engineered guide RNA comprises SEQ ID NO: 618.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/G mismatch at position -7 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 183 Embodiment 183.
  • Embodiment 182 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 662.
  • Embodiment 184 The composition of Embodiment 183, wherein the engineered guide RNA comprises SEQ ID NO: 662.
  • Embodiment 185 The composition of Embodiment 185.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 3 relative to position 0, a 2/2 symmetric bulge at position 7 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 186 Embodiment 186.
  • Embodiment 185 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 664.
  • Embodiment 187 The composition of Embodiment 186, wherein the engineered guide RNA comprises SEQ ID NO: 664.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/C mismatch at position -7 relative to position 0, a C/U mismatch at position -4 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 2/2 symmetric bulge at position 10 relative to position 0, an A/C mismatch at position 19 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 189 The composition of Embodiment 188, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 672 Embodiment 190.
  • the composition of Embodiment 189, wherein the engineered guide RNA comprises SEQ ID NO: 672.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, and any combination thereof.
  • Embodiment 192 The composition of Embodiment 191, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 675.
  • Embodiment 193 The composition of Embodiment 192, wherein the engineered guide RNA comprises SEQ ID NO: 675.
  • Embodiment 194. The composition of Embodiment 157, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/C mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/C mismatch at position 12 relative to position 0, a G/G mismatch at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, and any combination thereof.
  • Embodiment 198. The composition of Embodiment 197, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 699.
  • Embodiment 199 Embodiment 199.
  • Embodiment 198 wherein the engineered guide RNA comprises SEQ ID NO: 699.
  • Embodiment 200. The composition of Embodiment 157, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/A mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 201 The composition of Embodiment 200, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 700.
  • Embodiment 202 The composition of Embodiment 201, wherein the engineered guide RNA comprises SEQ ID NO: 700.
  • Embodiment 203 The composition of Embodiment 201, wherein the engineered guide RNA comprises SEQ ID NO: 700.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, and any combination thereof.
  • Embodiment 204 The composition of Embodiment 203, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 718.
  • Embodiment 205 The composition of Embodiment 204, wherein the engineered guide RNA comprises SEQ ID NO: 718.
  • Embodiment 206. The composition of Embodiment 157, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/C mismatch at position 5 relative to position 0, a C/A mismatch at position 9 relative to position 0, a C/A mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 207 The composition of Embodiment 206, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 719.
  • Embodiment 208. The composition of Embodiment 207, wherein the engineered guide RNA comprises SEQ ID NO: 719.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a 3/3 symmetric bulge at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/C mismatch at position 9 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 210 Embodiment 210.
  • composition of Embodiment 209, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 745.
  • the composition of Embodiment 210, wherein the engineered guide RNA comprises SEQ ID NO: 745.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 212 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 750.
  • Embodiment 214 The composition of Embodiment 213, wherein the engineered guide RNA comprises SEQ ID NO: 750.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a A/A mismatch at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a C/U mismatch at position 10 relative to position 0, a G/U Wobble at position 20 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 216 Embodiment 216.
  • Embodiment 215 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 762.
  • Embodiment 217 The composition of Embodiment 216, wherein the engineered guide RNA comprises SEQ ID NO: 762.
  • Embodiment 218 The composition of Embodiment 215, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 762.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a 3/3 symmetric bulge at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a U/U mismatch at position 5 relative to position 0, a G/U Wobble at position 8 relative to position 0, a C/U mismatch at position 14 relative to position 0, a G/G mismatch at position 17 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 219. The composition of Embodiment 218, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1099.
  • Embodiment 220. The composition of Embodiment 219, wherein the engineered guide RNA comprises SEQ ID NO: 1099.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -20 relative to position 0, a C/A mismatch at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a G/U Wobble at position 6 relative to position 0, a C/U mismatch at position 9 relative to position 0, a C/U mismatch at position 14 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 222 The composition of Embodiment 221, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1120. Embodiment 223. The composition of Embodiment 222, wherein the engineered guide RNA comprises SEQ ID NO: 1120. Embodiment 224.
  • composition of Embodiment 157 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof.
  • Embodiment 225 The composition of Embodiment 224, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1213.
  • Embodiment 226 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1213.
  • Embodiment 225 wherein the engineered guide RNA comprises SEQ ID NO: 1213.
  • the composition of Embodiment 157, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -22 relative to position 0, a 3/3 symmetric bulge mismatch at position -13 relative to position 0, a 6/6 symmetric internal loop at position -1 relative to position 0, a 3/3 symmetric bulge at position 38 relative to position 0, a 3/3 symmetric bulge at position 47 relative to position 0, a 3/3 symmetric bulge at position 56 relative to position 0, a 3/3 symmetric bulge at position 65 relative to position 0, and any combination thereof Embodiment 228.
  • Embodiment 227 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1214.
  • Embodiment 229. The composition of Embodiment 228, wherein the engineered guide RNA comprises SEQ ID NO: 1214 Embodiment 230.
  • composition of Embodiment 230 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -25 relative to position 0, a 3/3 symmetric bulge at position -16 relative to position 0, a 6/6 symmetric internal loop at position -4 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • Embodiment 232 Embodiment 232.
  • Embodiment 231 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 88.
  • Embodiment 233 The composition of Embodiment 232, wherein the engineered guide RNA comprises SEQ ID NO: 88.
  • composition of Embodiment 230 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • Embodiment 235 Embodiment 235.
  • Embodiment 234 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 903.
  • Embodiment 236 The composition of Embodiment 235, wherein the engineered guide RNA comprises SEQ ID NO: 903. Embodiment 237.
  • composition of Embodiment 230 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • Embodiment 238 Embodiment 238.
  • Embodiment 237 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1179.
  • Embodiment 239. The composition of Embodiment 238, wherein the engineered guide RNA comprises SEQ ID NO: 1179.
  • composition of Embodiment 230 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -4 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 37 relative to position 0, a 3/3 symmetric bulge at position 46 relative to position 0, a 3/3 symmetric bulge at position 55 relative to position 0, a 3/3 symmetric bulge at position 64 relative to position 0, and any combination thereof.
  • Embodiment 240 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1212. Embodiment 242.
  • composition of Embodiment 243 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -18 relative to position 0, a 6/6 symmetric internal loop at position -6 relative to position 0, a G/G mismatch at position - 1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 244 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 118.
  • Embodiment 246 The composition of Embodiment 245, wherein the engineered guide RNA comprises SEQ ID NO: 118.
  • Embodiment 247 The composition of Embodiment 244, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 118.
  • Embodiment 246 The composition of Embodiment 245, wherein the engineered guide RNA comprises SEQ ID NO: 118.
  • Embodiment 247 Embodiment 247.
  • composition of Embodiment 243 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -6 relative to position 0, a G/G mismatch at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 248 The composition of Embodiment 247, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 119.
  • Embodiment 249. The composition of Embodiment 248, wherein the engineered guide RNA comprises SEQ ID NO: 119.
  • Embodiment 250. The composition of Embodiment 243, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 250 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 270.
  • Embodiment 252. The composition of Embodiment 251, wherein the engineered guide RNA comprises SEQ ID NO: 270.
  • composition of Embodiment 243 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 254 The composition of Embodiment 253, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 271.
  • Embodiment 255 The composition of Embodiment 254, wherein the engineered guide RNA comprises SEQ ID NO: 271.
  • Embodiment 256. The composition of Embodiment 243, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -17 relative to position 0, a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 257 The composition of Embodiment 256, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 403.
  • Embodiment 258 The composition of Embodiment 257, wherein the engineered guide RNA comprises SEQ ID NO: 403.
  • composition of Embodiment 243 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 36 relative to position 0, a 3/3 symmetric bulge at position 45 relative to position 0, a 3/3 symmetric bulge at position 54 relative to position 0, a 3/3 symmetric bulge at position 63 relative to position 0, and any combination thereof.
  • Embodiment 260 The composition of Embodiment 259, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1081.
  • Embodiment 261 The composition of Embodiment 260, wherein the engineered guide RNA comprises SEQ ID NO: 1081.
  • Embodiment 262 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 23, relative to the target adenosine at position 0. Embodiment 263.
  • Embodiment 264 Embodiment 264.
  • Embodiment 263 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 401.
  • Embodiment 265. The composition of Embodiment 264, wherein the engineered guide RNA comprises SEQ ID NO: 401.
  • composition of Embodiment 262 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -5 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • Embodiment 267 The composition of Embodiment 266, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 402.
  • Embodiment 268 The composition of Embodiment 267, wherein the engineered guide RNA comprises SEQ ID NO: 402.
  • Embodiment 269. The composition of Embodiment 262, wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -19 relative to position 0, a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • Embodiment 269 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 458.
  • Embodiment 271. The composition of Embodiment 270, wherein the engineered guide RNA comprises SEQ ID NO: 458.
  • Embodiment 262 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -7 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 35 relative to position 0, a 3/3 symmetric bulge at position 44 relative to position 0, a 3/3 symmetric bulge at position 53 relative to position 0, a 3/3 symmetric bulge at position 62 relative to position 0, and any combination thereof.
  • Embodiment 273 The composition of Embodiment 272, wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 459.
  • Embodiment 274 The composition of Embodiment 273, wherein the engineered guide RNA comprises SEQ ID NO: 459.
  • Embodiment 275 The composition of Embodiment 10, wherein the first 6/6 symmetric internal loop is at position 22, relative to the target adenosine at position 0.
  • Embodiment 276 The composition of Embodiment 10 wherein the first 6/6 symmetric internal loop is at position 22, relative to the target adenosine at position 0.
  • composition of Embodiment 275 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 3/3 symmetric bulge at position -22 relative to position 0, a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 34 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • Embodiment 277 Embodiment 277.
  • Embodiment 276 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1135.
  • Embodiment 278 The composition of Embodiment 277, wherein the engineered guide RNA comprises SEQ ID NO: 1135.
  • composition of Embodiment 275 wherein the one or more structural features further comprises at least one structural feature selected from the group consisting of: a 6/6 symmetric internal loop at position -10 relative to position 0, a 2/2 symmetric bulge at position -1 relative to position 0, a 3/3 symmetric bulge at position 6 relative to position 0, a 3/3 symmetric bulge at position 34 relative to position 0, a 3/3 symmetric bulge at position 43 relative to position 0, a 3/3 symmetric bulge at position 52 relative to position 0, a 3/3 symmetric bulge at position 61 relative to position 0, and any combination thereof.
  • Embodiment 280 Embodiment 280.
  • Embodiment 279 wherein the engineered guide RNA comprises at least about: 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1136.
  • Embodiment 281. The composition of Embodiment 280, wherein the engineered guide RNA comprises SEQ ID NO: 1136.
  • Embodiment 282 The composition of any one of Embodiments 1-281, wherein the one or more structural features comprises at least a first 6/6 symmetric internal loop and at least a second 6/6 symmetric loop Embodiment 283.
  • the composition of any one of Embodiments 1-281, wherein the one or more structural features comprises the bulge, wherein the bulge is a symmetric bulge.
  • the composition of any one of Embodiments 1-281, wherein the one or more structural features comprises the bulge, wherein the bulge is an asymmetric bulge.
  • Embodiment 285. The composition of any one of Embodiments 1-284, wherein the one or more structural features comprises the internal loop, wherein the internal loop is a symmetric internal loop.
  • Embodiment 286 The composition of any one of Embodiments 1-284, wherein the one or more structural features comprises the internal loop, wherein the internal loop is an asymmetric internal loop.
  • Embodiment 287 The composition of any one of Embodiments 1-286, wherein the guide-target RNA scaffold comprises a Wobble base pair.
  • Embodiment 288 The composition of any one of Embodiments 1-287, wherein the one or more structural features comprises the hairpin, wherein the hairpin is a recruitment hairpin or a non-recruitment hairpin.
  • composition of any one of Embodiments 1-288 wherein upon hybridization of the engineered guide RNA to the sequence of the target MAPT RNA, the engineered guide RNA facilitates RNA editing of one or more adenosines in the sequence of the target MAPT RNA by an RNA editing entity.
  • RNA editing entity comprises ADAR1, ADAR2, ADAR3, or any combination thereof.
  • Embodiment 291 wherein the sequence of the MAPT RNA is the AUG in the 18th nucleotide in Exon 1 (c.1), and wherein the second sequence of the target MAPT RNA is selected from the group consisting of: an AUG in the 48th nucleotide in Exon 1 (c.31), an AUG in the 108th nt of Exon 1 (c.91), an AUG in the 6th nucleotide in Exon 5 (c.379), and any combination thereof.
  • Embodiment 293. The composition of any one of Embodiments 1-292, wherein the engineered guide RNA is encoded by an engineered polynucleotide.
  • Embodiment 293 wherein the engineered polynucleotide is comprised in or on a vector.
  • Embodiment 295. The composition of Embodiment 294, wherein the vector is a viral vector, and wherein the engineered polynucleotide is encapsidated in the viral vector.
  • Embodiment 296. The composition of Embodiment 295, wherein the viral vector is an adeno-associated viral (AAV) vector or a derivative thereof.
  • AAV adeno-associated viral
  • Embodiment 295 wherein the viral vector is an adeno-associated viral (AAV) vector and wherein the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a derivative, a chimera, or a variant of any of these.
  • Embodiment 298 The composition of any one of Embodiments 296-297, wherein the AAV vector is a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, or any combination thereof.
  • Embodiment 301. The composition of any one of Embodiments 1-300, in unit dose form.
  • a pharmaceutical composition comprising: the composition of any one of Embodiments 1-301; and a pharmaceutically acceptable: excipient, carrier, or diluent.
  • Embodiment 303 The pharmaceutical composition of Embodiment 302, in unit dose form.
  • Embodiment 304. A method of treating a disease or a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of Embodiments 1-301 or the pharmaceutical composition of Embodiment 302 or 303.
  • Embodiment 305. The method of Embodiment 304, wherein the disease or condition comprises a Tauopathy.
  • Embodiment 304 wherein the disease or condition comprises Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy, autism, traumatic brain injury, or Dravet syndrome.
  • Embodiment 307. The method of any one of Embodiments 304-306, wherein the subject is a human or a non-human animal.
  • Embodiment 308. The method of any one of Embodiments 304-307, wherein the administering is sufficient to treat one or more symptoms of the disease of condition.
  • Embodiment 308 wherein the one of more symptoms treated comprises a reduction in memory loss, a reduction in confusion, or any combination thereof, as compared to prior to the administering.
  • Embodiment 310 The method of Embodiment 308-309, wherein the administering is sufficient to reduce aggregation of tau protein, relative to: a level of aggregation prior to the administering; a level of accumulated aggregation in the subject in the absence of the administering; or both.
  • Embodiment 311 The method of Embodiment 308-309, wherein the administering is sufficient to reduce an amount of: neurofibrillary tangles; neuronal cell death; neuroinflammation; or any combination thereof; relative to an amount prior to the administering.
  • Embodiment 312 The composition of Embodiment 308, wherein the amount of phosphorylated tau protein is decreased in the subject after the administering, relative to an amount of phosphorylated tau protein prior to the administering.
  • Embodiment 313. The composition of Embodiment 308, wherein a ratio of a 4R isoform of Tau to a 3R isoform of tau is increased upon administration, relative to a ratio prior to the administering.
  • Embodiment 314. The composition of any one of Embodiments 1-301 or the pharmaceutical composition of Embodiment 302 or 303 for use in treatment of a disease or condition.
  • Embodiment 315 The composition or the pharmaceutical composition for use of Embodiment 314, wherein the disease or condition comprises a Tauopathy.
  • Embodiment 316 The composition or the pharmaceutical composition for use of Embodiment 314, wherein the disease or condition comprises Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy, autism, traumatic brain injury, or Dravet syndrome.
  • Embodiment 317 A method of editing a MAPT RNA, the method comprising contacting the MAPT RNA with the composition of any one of Embodiments 1-301 and an RNA editing entity, thereby editing the MAPT RNA.
  • Embodiment 318 A method of editing a MAPT RNA, the method comprising contacting the MAPT RNA with the composition of any one of Embodiments 1-301 and an RNA editing entity, thereby editing the MAPT RNA.
  • Embodiment 317 wherein the editing comprises editing one or more adenosines within the 3’ untranslated region (UTR) of the MAPT RNA.
  • Embodiment 319 The method of Embodiment 317, wherein the editing comprises editing one or more adenosines within the 5’ untranslated region (UTR) of the MAPT RNA.
  • Embodiment 320 The method of Embodiment 317, wherein the editing comprises editing one or more adenosines of a transcription initiation site (TIS) of the MAPT RNA.
  • TIS transcription initiation site
  • Embodiment 320 wherein the translation initiation site is the c.1 TIS corresponding to the canonical TIS at nucleotide position 151 of the MAPT reference transcript of NM_001377265.1.
  • Embodiment 322. The method of any one of Embodiments 320-321, wherein the MAPT RNA comprises a pre-mRNA transcript of MAPT.
  • Embodiment 323. The method of Embodiment 322, wherein at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the pre-mRNA transcripts of MAPT have at least one edit.
  • Embodiment 317 wherein the editing of MAPT RNA facilitates a protein knockdown.
  • Embodiment 325 The method of Embodiment 324, wherein the protein knockdown comprises a reduction of at least 10%, relative to an amount of protein present prior to the contacting.
  • Embodiment 326 The method of Embodiment 324, wherein the protein knockdown comprises a reduction of from about 10% to about 25%, relative to an amount of protein present prior to the contacting.
  • Embodiment 327 The method of Embodiment 324, wherein the protein knockdown comprises a reduction of at least 50%, relative to an amount of protein present prior to the contacting.
  • Embodiment 328 The method of Embodiment 317, wherein the editing of MAPT RNA facilitates a protein knockdown.
  • Embodiment 324 wherein the protein knockdown comprises a knockdown of tau Embodiment 329.
  • the method of any one of Embodiments 324-328, wherein the knockdown is measured in an in vitro assay.
  • Embodiment 330 The method of any one of Embodiments 324-328, wherein the knockdown is measured in an in vivo assay.
  • Embodiment 331 The method of any one of Embodiments 324- 328, wherein the knockdown is measured in a human subject.
  • Engineered Guide RNAs for Editing MAPT TIS This example describes engineered guide RNAs for editing MAPT RNA to knockdown expression of the Tau protein.
  • Engineered guide RNAs of the present disclosure are designed to target a translation initiation site (TIS) of MAPT (e.g., an AUG in the 18th nt in Exon 1 (c.1), an AUG in the 48 th nt in Exon 1 (c.31), an AUG in the 108 th nt of Exon 1 (c.91), an AUG in the 6 th nt in Exon 5 (c.379), or any combination thereof) RNA and facilitate ADAR-mediated RNA editing of AUG (the TIS) to GUG, thus, inhibiting MAPT translation.
  • TIS translation initiation site
  • Engineered guide RNAs are packaged and delivered in AAV virus and are administered to a subject in need thereof.
  • the engineered guide RNAs edit the MAPT TIS, thereby reducing Tau protein expression.
  • the engineered guide RNAs are therapeutically effective and reduce symptoms and/or cure the Tauopathy.
  • RNA editing entity e.g., a recombinant ADARl and/or ADAR2
  • each structural feature formed within a guide-target RNA scaffold is annotated as follows: a. the position of the structural feature with respect to the target A (position 0) of the target RNA sequence, with a negative value indicating upstream (5’) of the target A and a positive value indicating downstream (3’) of the target A; b. the number of bases in the target RNA sequence and the number of bases in the engineered guide RNA that together form the structural feature - for example, 6/6 indicates that six contiguous bases from the target RNA sequence and six contiguous bases from the engineered guide RNA form the structural feature; c.
  • the name of the structural feature e.g., symmetric bulge, symmetric internal loop, asymmetric bulge, asymmetric internal loop, mismatch, or wobble base pair
  • the sequences of bases on the target RNA side and the engineered guide RNA side that participate in forming the structural feature e.g., symmetric bulge, symmetric internal loop, asymmetric bulge, asymmetric internal loop, mismatch, or wobble base pair
  • “-22 3-3 bulge-symmetric CUA-GAU” is read as a structural feature formed in a guide-target RNA scaffold (target MAPT c.1 TIS RNA sequence hybridized to an engineered guide RNA of SEQ ID NO: 2), where a. the structural feature starts 22 nucleotides upstream (5’) (the -22 position) from the target A (0 position) of the target RNA sequence b. three contiguous bases from the target RNA sequence and three contiguous bases from the engineered guide RNA form the structural feature c. the structural feature is a symmetric bulge d. a sequence of CUA from the target RNA side and a sequence of GAU from the engineered guide RNA side participate in forming the symmetric bulge.
  • FIG. 1 can be used as an aid to visualize the structural features and the nomenclature disclosed herein.
  • FIG. 2 is a plot showing, on the x-axis, the sequence similarity of the MAPT c.l TIS-targeting engineered guide RNAs of the present disclosure to a canonical guide RNA design and, on the y-axis, the edited fraction by an ADAR2 enzyme.
  • Table 1 further includes the amount of on target editing achieved via ADARl or ADAR2 seperately, as well as ADARl and ADAR2. The specificity of each guide was also calculated for each engineered guide via ADARl, ADAR2, and ADARl +ADAR2.

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Abstract

L'invention concerne des ARN guides modifiés et des compositions les comprenant pour le traitement de maladies ou d'états chez un sujet. L'invention concerne également des procédés de traitement de maladies ou d'états chez un sujet par administration d'ARN guides modifiés ou de compositions pharmaceutiques selon l'invention.
EP22748526.5A 2021-06-29 2022-06-28 Arn guides et polynucléotides modifiés Pending EP4363576A1 (fr)

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US20170211064A1 (en) * 2014-07-29 2017-07-27 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating tau expression
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